ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection

  1. Asmaa Hachim
  2. Niloufar Kavian
  3. Carolyn A. Cohen
  4. Alex W. H. Chin
  5. Daniel K. W. Chu
  6. Chris K. P. Mok
  7. Owen T. Y. Tsang
  8. Yiu Cheong Yeung
  9. Ranawaka A. P. M. Perera
  10. Leo L. M. Poon
  11. J. S. Malik Peiris
  12. Sophie A. Valkenburg

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Abstract

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  1. Version published to 10.1038/s41590-020-0773-7
  2. ScreenIT

    SciScore for 10.1101/2020.04.30.20085670: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Fifteen patients were enrolled in Hong Kong (China, SAR) and all of them provided informed consent.
    IRB: The study was approved by the institutional review board of the Hong Kong West Cluster of the Hospital Authority of Hong Kong (approval number:
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    SARS-CoV-2 gene cloning: Based on previous studies describing the structure of the SARS-CoV-2 genome (1, 8), an extensive panel of 12 proteins (S, E, M, N, NSP1, ORF3a, 3b, 6, 7a, 7b, 8, 10) was chosen for antibody testing by LIPS.
    SARS-CoV-2 genome ( 1 , 8
    suggested: None
    NSP1
    suggested: None
    Following FBS blocking and thorough washing, diluted plasma samples (1:100) were bound for 2 hours, further washed and then detected by an anti-human Ig secondary antibody labelled with HRP specific for IgG (Invitrogen, Carlsbad, CA, USA).
    anti-human Ig secondary antibody labelled with HRP specific for IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    After 1 hour of incubation at 37°C, 35µl of the virus-serum mixture was added in quadruplicate to Vero or Vero E6 cell monolayers in 96-well microtiter plates.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Multiple alignments of Coronaviruses: Multiple amino acid alignments of structural proteins from HKU1 (AY597011.2), HCoV-229E (AF304460.1), HCoV-OC43 (AY391777.1) and HCoV-NL63 (AY567487.2) were compared versus SARS-CoV-2 (MN908947) using CLUSTAL 2.1.
    CLUSTAL
    suggested: (Clustal X , RRID:SCR_017055)
    : GraphPad Prism 6 software (San Diego, CA) was used for statistical analysis.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Non-parametric Mann–Whitney U tests were used to compare the antibody levels between COVID-19 and negative groups, using the GraphPad 8 Prism software.
    GraphPad
    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:
    The absence of paired samples from multiple time-points for the same patients is also a limitation. Longitudinal studies are needed to further assess kinetics of antibody responses, especially to identify early antibody responses for rapid diagnostic tests. The inclusion of additional time points, greater sample size and asymptomatic cases are necessary to test the cut-off sensitivity scores; and to confirm the diagnostic value of our LIPS assays and the relevancy of ORF3b and ORF8 antibodies. Late-convalescent sera are needed to assess antibody waning. We also need to investigate antibody profiles with disease severity. In conclusion, we found that COVID-19 patients not only produce antibodies to the Spike protein, but also to other structural and non-structural proteins. The nucleoprotein, and th en ORF8, and ORF3 show an immunodominant and specific humoral response compared with other SARS-CoV-2 antigenic targets tested. The combined use of N+ORF3b+ORF8 provides a sensitive and specific method for the detection of all COVID-19 patients in our cohort even at early time-points, whilst the Spike protein does not. Our results provide insights into the overall spectrum of antibody responses associated with COVID-19. We still need to investigate whether antigens other than the virus spike confer protection. Such information will help prioritize antigen targets for vaccine development, monoclonal antibody reagents and detecting early responses to infection.

    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.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.04.30.20085670v1 on medRxiv