Dataset Information

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

ABSTRACT:

Introduction

Evaluation 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.

Methods

Enzyme-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).

Results

The 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.

Conclusions

In 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.

SUBMITTER: Villemonteix J

PROVIDER: S-EPMC9449588 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

Villemonteix Juliette J Cohen Laure L Guihot Amélie A Guérin Valérie V Moulin Clémentine C Caseris Marion M Carol Agnès A Bonacorsi Stéphane S Carcelain Guislaine G

Immunity, inflammation and disease 20221001 10

<h4>Introduction</h4>Evaluation 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.<h4>Methods</h4>Enzyme-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).<h4>Results</h4>The proportion o ...[more]

PMID: 36169252

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Project description:BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Project description:BackgroundEbola virus (EBOV) caused more than 11,000 deaths during the 2013-2016 epidemic in West Africa without approved vaccines or immunotherapeutics. Despite its high lethality in some individuals, EBOV infection can produce little to no symptoms in others. A better understanding of the immune responses in individuals who experienced minimally symptomatic and asymptomatic infection could aid the development of more effective vaccines and antivirals against EBOV and related filoviruses.Methodology/principle findingsBetween August and November 2017, blood samples were collected from 19 study participants in Lagos, Nigeria, including 3 Ebola virus disease (EVD) survivors, 10 individuals with documented close contact with symptomatic EVD patients, and 6 control healthcare workers for a cross-sectional serosurvey and T cell analysis. The Lagos samples, as well as archived serum collected from healthy individuals living in surrounding areas of the 1976 Democratic Republic of Congo (DRC) epidemic, were tested for EBOV IgG using commercial enzyme-linked immunosorbent assays (ELISAs) and Western blots. We detected antibodies in 3 out of 3 Lagos survivors and identified 2 seropositive individuals not known to have ever been infected. Of the DRC samples tested, we detected antibodies in 9 out of 71 (12.7%). To characterize the T cell responses in the Lagos samples, we developed an anthrax toxin-based enzyme-linked immunospot (ELISPOT) assay. The seropositive asymptomatic individuals had T cell responses against EBOV nucleoprotein, matrix protein, and glycoprotein 1 that were stronger in magnitude compared to the survivors.Conclusion/significanceOur data provide further evidence of EBOV exposure in individuals without EVD-like illness and, for the first time, demonstrate that these individuals have T cell responses that are stronger in magnitude compared to severe cases. These findings suggest that T cell immunity may protect against severe EVD, which has important implications for vaccine development.

Project description:BackgroundQualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity.MethodsWhole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period.ResultsThe limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells.ConclusionsThe whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Dataset Information

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

Introduction

Methods

Results

Conclusions

Publications

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

Similar Datasets

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