The β-NGF/TrkA Signalling Pathway Is Associated With the Production of Anti-Nucleoprotein IgG in Convalescent COVID-19

  1. Carla Usai
  2. Joseph M. Gibbons
  3. Corinna Pade
  4. Wenhao Li
  5. Sabina R. M. Jacobs
  6. Áine McKnight
  7. Patrick T. F. Kennedy
  8. Upkar S. Gill

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Abstract

The presentation of SARS-CoV-2 infection varies from asymptomatic to severe COVID-19. Similarly, high variability in the presence, titre and duration of specific antibodies has been reported. While some host factors determining these differences, such as age and ethnicity have been identified, the underlying molecular mechanisms underpinning these differences remain poorly defined.

Methods

We analysed serum and PBMC from 17 subjects with a previous PCR-confirmed SARS-CoV-2 infection and 10 unexposed volunteers following the first wave of the pandemic, in the UK. Anti-NP IgG and neutralising antibodies were measured, as well as a panel of infection and inflammation related cytokines. The virus-specific T cell response was determined by IFN-γ ELISPOT and flow cytometry after overnight incubation of PBMCs with pools of selected SARS-CoV-2 specific peptides.

Results

Seven of 17 convalescent subjects had undetectable levels of anti-NP IgG, and a positive correlation was shown between anti-NP IgG levels and the titre of neutralising antibodies (IC50). In contrast, a discrepancy was noted between antibody levels and T cell IFN-γ production by ELISpot following stimulation with specific peptides. Among the analysed cytokines, β-NGF and IL-1α levels were significantly different between anti-NP positive and negative subjects, and only β-NGF significantly correlated with anti-NP positivity. Interestingly, CD4 + T cells of anti-NP negative subjects expressed lower amounts of the β-NGF-specific receptor TrkA.

Conclusions

Our results suggest that the β-NGF/TrkA signalling pathway is associated with the production of anti-NP specific antibody in mild SARS-CoV-2 infection and the mechanistic regulation of this pathway in COVID-19 requires further investigation.

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  1. Version published to 10.3389/fimmu.2021.813300
  2. ScreenIT

    SciScore for 10.1101/2021.11.11.21266223: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All participants provided informed consent according to the local ethics committee approval
    IRB: All participants provided informed consent according to the local ethics committee approval
    Sex as a biological variablenot detected.
    RandomizationConvalescent COVID-19 and Healthy Donors: Forty donors were randomly selected from a previously published cohort (2) to create four sex- and age-matched groups according to PCR and antibody status.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody tests: The presence of anti-Nucleocapsid protein (NP) IgG and IgM in serum samples was determined using the Panbio™ COVID-19 IgG/IgM Rapid Test Device (Fingerstick Whole Blood/Venous Whole Blood/Serum/Plasma) (Panbio™; Abbott Rapid Diagnostics Jena GmbH, Jena, Germany) according to the manufacturer’s instructions and as previously described (2).
    anti-Nucleocapsid protein ( NP ) IgG
    suggested: None
    Each well was incubated for 20 min at 4°C in the dark, with saturating concentrations (100 µl) of a mix of the following antibodies: anti-PD1 PE-Cy7 (BioLegend, clone EH12.2H7)
    anti-PD1 PE-Cy7
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    VeroE6 cells were seeded in 96-well plates 24h prior to infection.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Anti-NPIgG leves in serum samples were quantified using the Abbott Architect i2000 chemiluminescent microparticle immunoassay (Architect) was for SARS-CoV-2 IgG (Abbott Diagnostics, IL, USA; Architect) according to the manufacturer’s instructions and as previously described (2).
    Abbott Architect
    suggested: (Abbott ARCHITECT i1000sr System, RRID:SCR_019328)
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    Cells were acquired on a BD-LSR II FACS Scan, and data were analysed using FlowJo (Tree Star Inc.).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Statistical analysis was performed using GraphPad Prism 9.1.2,
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Software, San Diego, California USA, (
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Binary logistic regression was performed using IBM SPSS Statistics for Windows, Version 27.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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:
    We acknowledge the limitations of our study, in part linked to the difficulties in diagnosis during the early phase of the pandemic, when PCR testing was not widely available. For this reason, we were not able to determine the exact timeframe between symptom onset, PCR test and sample collection for most of our subjects. Moreover, our sample size was limited, especially the group of PCR positive anti-NP negative subjects; this could be considered an intrinsic limitation of this research field, since only 1-10% of PCR confirmed cases are estimated to have undetectable antibodies. In addition, we acknowledge the fact that only four out of seven anti-NP negative, PCR positive subjects in our cohort had given their consent for PBMC isolation from venous blood. The ex-vivo assays were not performed using overlapping peptides covering the entire sequence of the SARS-CoV-2 proteins, but only selected peptides were used; however, since they were found to be immunogenic (17,27) we are confident that the results we obtained are representative, and are in line with other studies reviewed by Bertoletti et al.(30). We describe for the first time the β-NGF/TrkA signalling pathway as a host factor reflecting different levels of inflammation within mild COVID-19 cases, with effects on the virus-specific humoral and T cell response. The mechanistic regulation of this pathway in COVID-19 disease deserves further investigation, and larger studies are required to determine whether the effects of...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    ISRCTN60400862NANA

    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.

    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.

    About SciScore

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  3. Version published to 10.1101/2021.11.11.21266223v1 on medRxiv