T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses
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Abstract
Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.
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SciScore for 10.1101/2020.09.28.20202929: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics Statement: Human study protocols were approved by the research ethics committee (REC) at Yorkshire & The Humber - Sheffield (GI Biobank Study 16/YH/0247).
Consent: Written informed consent was obtained for all patients enrolled in the study.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells from proliferation assays were incubated with fluorochrome-conjugated primary human-specific antibodies for CD3, CD4 and CD8 in cell staining buffer (Biolegend) containing serum for 30min at 4°C, washed with cell … SciScore for 10.1101/2020.09.28.20202929: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethics Statement: Human study protocols were approved by the research ethics committee (REC) at Yorkshire & The Humber - Sheffield (GI Biobank Study 16/YH/0247).
Consent: Written informed consent was obtained for all patients enrolled in the study.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells from proliferation assays were incubated with fluorochrome-conjugated primary human-specific antibodies for CD3, CD4 and CD8 in cell staining buffer (Biolegend) containing serum for 30min at 4°C, washed with cell staining buffer, fixed with 4% paraformaldehyde (PFA, Sigma) and stored at 4°C in the dark until data acquisition. CD3suggested: NoneCD4suggested: NoneCD8suggested: NoneStandardised ELISA for detection of SARS-CoV-2 spike antigen-specific total IgG in plasma: Total anti-SARS CoV-2 spike antibodies were determined using an indirect ELISA as described previously22, which is based on the Krammer assay34 using a standard curve derived from a pool of SARS-COV-2 convalescent plasma samples on every plate. antigen-specific total IgGsuggested: Noneanti-SARSsuggested: NoneBriefly, neutralising antibody titres were determined by incubating serial two-fold plasma dilutions with ∼10^5 RLU pseudotyped virus for 2hrs before addition of 10^4 HEK293T cells transfected with full-length human ACE2 24hrs prior. ACE2suggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, neutralising antibody titres were determined by incubating serial two-fold plasma dilutions with ∼10^5 RLU pseudotyped virus for 2hrs before addition of 10^4 HEK293T cells transfected with full-length human ACE2 24hrs prior. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Experimental Models: Organisms/Strains Sentences Resources Briefly, 96-well Multiscreen-I plates (Millipore, UK) were coated for 3 hours with 10 μg/ml GZ-4 anti-human IFN-γ (Mabtech, AB, Sweden) at room temperature. ABsuggested: RRID:BDSC_203)Software and Algorithms Sentences Resources Data was acquired on an LSRII flow cytometer (BD Biosciences) and analysis was performed with FlowJo Version 10 (BD Biosciences). FlowJosuggested: (FlowJo, RRID:SCR_008520)Standardised EUs were determined from a single dilution of each sample against the standard curve which was plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek). Gen5suggested: (Gen5, RRID:SCR_017317)), readouts were normalised, and -Log(IC50) determined via non-linear regression using GraphPad Prism8 GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Statistical analyses: Statistical analysis was performed with IBM SPSS Statistics 25 and figures were made with GraphPad Prism 8. SPSSsuggested: (SPSS, RRID:SCR_002865)GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)For polyfunctionality analyses, data was prepared using PESTEL v2.0 for formatting and baseline subtraction, followed by export of data to SPICE v6.0 for analysis. SPICEsuggested: (SPICE, RRID:SCR_016603)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.09.28.20202929: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement MATERIALS AND METHODS Ethics Statement Human study protocols were approved by the research ethics committee (REC) at Yorkshire & The Humber - Sheffield (GI Biobank Study 16/YH/0247). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells from proliferation assays were incubated with fluorochromeconjugated primary human-specific antibodies for CD3, CD4 and CD8 in cell staining buffer (Biolegend) containing serum for 30min at 4⁰C, washed with cell staining buffer, fixed with 4% paraformaldehyde (PFA, Sigma) and stored … SciScore for 10.1101/2020.09.28.20202929: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement MATERIALS AND METHODS Ethics Statement Human study protocols were approved by the research ethics committee (REC) at Yorkshire & The Humber - Sheffield (GI Biobank Study 16/YH/0247). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells from proliferation assays were incubated with fluorochromeconjugated primary human-specific antibodies for CD3, CD4 and CD8 in cell staining buffer (Biolegend) containing serum for 30min at 4⁰C, washed with cell staining buffer, fixed with 4% paraformaldehyde (PFA, Sigma) and stored at 4⁰C in the dark until data acquisition. CD3suggested: NoneCD4suggested: NoneCD8suggested: NoneStandardised ELISA for detection of SARS-CoV-2 spike antigen-specific total IgG in plasma Total anti-SARS CoV-2 spike antibodies were determined using an indirect ELISA as described previously22, which is based on the Krammer assay34 using a standard curve derived from a pool of SARS-COV-2 convalescent plasma samples on every plate. antigen-specific total IgGsuggested: Noneanti-SARSsuggested: NoneBriefly, neutralising antibody titres were determined by incubating serial two-fold plasma dilutions with ~10^5 RLU pseudotyped virus for 2hrs before addition of 10^4 HEK293T cells transfected with full-length human ACE2 24hrs prior. ACE2suggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, neutralising antibody titres were determined by incubating serial two-fold plasma dilutions with ~10^5 RLU pseudotyped virus for 2hrs before addition of 10^4 HEK293T cells transfected with full-length human ACE2 24hrs prior. HEK293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Experimental Models: Organisms/Strains Sentences Resources Briefly, 96-well Multiscreen-I plates (Millipore, UK) were coated for 3 hours with 10 μg/ml GZ-4 anti-human IFN-γ (Mabtech, AB, Sweden) at room temperature. ABsuggested: RRID:BDSC_203)Software and Algorithms Sentences Resources Data was acquired on an LSRII flow cytometer (BD Biosciences) and analysis was performed with FlowJo Version 10 (BD Biosciences). FlowJosuggested: (FlowJo, RRID:SCR_008520)Standardised EUs were determined from a single dilution of each sample against the standard curve which was plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek). Gen5suggested: (Gen5, RRID:SCR_017317)), readouts were normalised, and -Log(IC50) determined via non-linear regression using GraphPad Prism8 GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Statistical analyses Statistical analysis was performed with IBM SPSS Statistics 25 and figures were made with GraphPad Prism 8. SPSSsuggested: (SPSS, RRID:SCR_002865)GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)For polyfunctionality analyses, data was prepared using PESTEL v2.0 for formatting and baseline subtraction, followed by export of data to SPICE v6.0 for analysis. SPICEsuggested: (SPICE, RRID:SCR_016603)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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