A high content microscopy-based platform for detecting antibodies to the nucleocapsid, spike and membrane proteins of SARS-CoV-2

  1. Daniel M. Williams
  2. Hailey Hornsby
  3. Ola M. Shehata
  4. Rebecca Brown
  5. Domen Zafred
  6. Amber S.M. Shun-Shion
  7. Anthony J. Hodder
  8. Deepa Bliss
  9. Andrew Metcalfe
  10. James R. Edgar
  11. David E. Gordon
  12. Jon R. Sayers
  13. Martin J. Nicklin
  14. Paul J. Collini
  15. Steve Brown
  16. Thushan I. de Silva
  17. Andrew A. Peden

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Abstract

The strong humoral immune response produced against the SARS-CoV-2 nucleocapsid (N) and spike (S) proteins has underpinned serological testing but the prevalence of antibody responses to other SARS-CoV-2 proteins, which may be of use as further serological markers, is still unclear. Cell-based serological screening platforms can fulfil a crucial niche in the identification of antibodies which recognise more complex folded epitopes or those incorporating post-translation modifications which may be undetectable by other methods used to investigate the antigenicity of the SARS-CoV-2 proteome. Here, we employed automated high content immunofluorescence microscopy (AHCIM) to assess the viability of such an approach as a method capable of assaying humoral immune responses against full length SARS-CoV-2 proteins in their native cellular state. We first demonstrate that AHCIM provides high sensitivity and specificity in the detection of SARS-CoV-2 N and S IgG. Assessing the prevalence of antibody responses to the SARS-CoV-2 structural membrane protein (M), we further find that 85% of COVID-19 patients within our sample set developed detectable M IgG responses (M sensitivity 85%, N sensitivity 93%, combined N + M sensitivity 95%). The identification of M as a serological marker of high prevalence may be of value in detecting additional COVID-19 cases during the era of mass SARS-CoV-2 vaccinations, where serological screening for SARS CoV-2 infections in vaccinated individuals is dependent on detection of antibodies against N. These findings highlight the advantages of using cell-based systems as serological screening platforms and raise the possibility of using M as a widespread serological marker alongside N and S.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.14.21264873: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and plasmids: For immunofluorescence microscopy experiments StrepTagged proteins were detected using StrepTactin-DY549 (1:1000, IBA Lifesciences 2-1565-050) and human IgG with a rabbit anti-human-IgG AlexaFluor-488 (1:500, Life technologies A1101).
    StrepTactin-DY549
    suggested: None
    anti-human-IgG
    suggested: None
    Primary antibodies used for western blotting were mouse anti-SARS-CoV-2 nucleocapsid (Genetex, GTX632269), mouse anti-strep-mAb (1:1000, IBA Lifesciences) and rabbit anti-GAPDH (Proteintech, 60004-1-Ig).
    anti-SARS-CoV-2 nucleocapsid (Genetex, GTX632269)
    suggested: None
    anti-strep-mAb
    suggested: None
    anti-GAPDH
    suggested: None
    Secondary antibodies for western blotting were HRP conjugated donkey anti-human IgG (1:2000, Biolegend 410902), HRP conjugated anti-mouse (1:2000, Jackson Immuno Research 115-035-008) and HRP-conjugated goat anti-rabbit (1:2000, Jackson Immuno Research 111-035-144).
    anti-mouse
    suggested: (Jackson ImmunoResearch Labs Cat# 115-035-008, RRID:AB_2313585)
    anti-rabbit
    suggested: (Jackson ImmunoResearch Labs Cat# 111-035-144, RRID:AB_2307391)
    Bound antibodies and StrepTagged viral proteins were detected by incubating the coverslips with an anti-human IgG secondary antibody conjugated to AlexaFluor-488 (1:500, Life technologies A11014) and StrepTactin-DY549 (1:1000, IBA lifesciences 2-1565-050, 1 in 500 in IF buffer) for 1 hour at room temperature.
    anti-human IgG
    suggested: (Fitzgerald Industries International Cat# 61R-I167, RRID:AB_10814124)
    Of note, our initial experiments assaying IgG responses against N suggested patient sera N antibody levels were limiting as we observed that having too many transfected cells significantly reduced the number of virus specific antibodies bound to transfected cells, as indicated by a weaker anti-human IgG-1 Alexa-488 signal at higher transfected cell densities (Supplementary Figure 1).
    anti-human IgG-1
    suggested: None
    Membranes were then probed with either patient sera diluted 1:1000 in blocking solution or an anti-StrepTag monoclonal antibody (1:1000, IBA lifesciences 2-1507-001) overnight or for 1 hour at room temperature.
    anti-StrepTag
    suggested: None
    Membranes were washed three times for 5 minutes each with PBST and incubated with either donkey anti-human (1:2000, Biolegend 410902) or anti-mouse (1:2000, Jackson Immuno Research 115-035-008) HRP conjugated secondary antibodies for a further 1 hour.
    anti-human (1:2000, Biolegend 410902
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Automated high content microscopy and image analysis: HEK cells seeded in 6 well plates were transfected with SARS-CoV-2 plasmid DNA encoding either S, N or M proteins or mock transfected as described above, Cells were detached by rinsing with conditioned medium 24 hours after transfection.
    HEK
    suggested: None
    Western blotting: HEK-293T cells expressing the nucleocapsid protein grown in 6 or 12 well dishes were detached by rinsing with conditioned media and cell suspensions collected in 15ml falcon tubes.
    HEK-293T
    suggested: None
    Recombinant DNA
    SentencesResources
    All SARS-CoV-2 proteins were human codon optimised and expressed from pLVX-EF1alpha-IRES-puro (N and M) or pTwist-EF1alpha-IRES-puro (S) plasmids as previously described (Gordon, Jang et al. 2020).
    pLVX-EF1alpha-IRES-puro
    suggested: None
    pTwist-EF1alpha-IRES-puro
    suggested: None
    Automated high content microscopy and image analysis: HEK cells seeded in 6 well plates were transfected with SARS-CoV-2 plasmid DNA encoding either S, N or M proteins or mock transfected as described above, Cells were detached by rinsing with conditioned medium 24 hours after transfection.
    SARS-CoV-2
    suggested: RRID:Addgene_164583)
    Software and Algorithms
    SentencesResources
    Statistical comparisons between groups using an unpaired t-test and pearsons correlation coefficient analysis of N, S and M antibody relationships were all performed using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    With these caveats in mind, complementation of ELISA based detection of N antibodies with detection of M antibodies by AHCIM could provide an immediate strategy to enhance the sensitivity and specificity of SARS-CoV-2 serological testing during the era of mass COVID-19 vaccinations. Future work to identify the key immunodominant epitopes of M detected by AHCIM may simplify serological testing using M even further, allowing the production of a SARS-CoV-2 M protein compatible for use with an ELISA format, thereby helping to facilitate widespread serological testing using M. Another key issue in SARS-CoV-2 serology relates to the length of time that each serological marker persists for post infection (Meyer 2021). Long lived serological markers are desirable for optimal COVID-19 sero-surveillance as the loss of detectable antibody levels after infection may lead to an underestimation of infection rates and indicate a loss of protective immunity. Whilst detection of sero-conversion due to SARS-CoV-2 infection in those vaccinated is reliant on assaying for N antibodies, the utility of N as a long-lived serological marker is uncertain (Long, Tang et al. 2020, Ripperger, Uhrlaub et al. 2020, Fenwick, Croxatto et al. 2021, Whitcombe, McGregor et al. 2021), although recent data suggests that N antibodies levels remain high for longer than previously believed (Shrotri, Harris et al. 2021). Elucidating the kinetics of M antibody levels over time may help to establish whether M may be a ...

    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.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2021.10.14.21264873 on medRxiv