SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients

  1. Mohau S. Makatsa
  2. Marius B. Tincho
  3. Jerome M. Wendoh
  4. Sherazaan D. Ismail
  5. Rofhiwa Nesamari
  6. Francisco Pera
  7. Scott de Beer
  8. Anura David
  9. Sarika Jugwanth
  10. Maemu P. Gededzha
  11. Nakampe Mampeule
  12. Ian Sanne
  13. Wendy Stevens
  14. Lesley Scott
  15. Jonathan Blackburn
  16. Elizabeth S. Mayne
  17. Roanne S. Keeton
  18. Wendy A. Burgers

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Abstract

Background : The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings.

Methods : We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana . We measured antibody responses in sera from South African patients ( n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma ( n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva.

Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers.

Conclusion : We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

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  1. Version published to 10.3389/fpls.2021.589940
  2. ScreenIT

    SciScore for 10.1101/2020.08.04.20167940: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethical approval for these studies was obtained from the Human Research Ethics Committee (HREC) of the University of Witwatersrand (M200468) and the University of Cape Town (UCT; 210/2020).
    Consent: All participants provided written, informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analyses were performed in Prism (GraphPad, version 8).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    AUC was calculated in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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:
    A limitation of our study was that we did not have information on the date of COVID-19 symptom onset in our cohort, limiting our analyses to time post PCR positivity, which did not yield a relationship with antibody positivity or OD value. Additional factors that may also influence antibody generation and kinetics include disease severity, age and comorbidities. We found no relationship between increasing disease severity and antibody positivity or OD value, likely due to the fact that the majority of our study participants had mild to moderate COVID-19. We determined endpoint titers of binding antibodies to S1 and RBD in a subset of 20 convalescent participants in our cohort. Several studies have demonstrated that binding antibody titers against S correlate with neutralization capacity (Amanat et al., 2020; Okba et al., 2020; Premkumar et al., 2020). A recent study reporting S-specific IgG titers in almost 20 000 patients screened for eligibility as convalescent plasma donors demonstrated that 70% of IgG+ donors had high titers (>1:960) of antibodies (Wajnberg et al., 2020). Importantly, 100% of those with titers >2880 exhibited neutralizing activity (ID50 of >1:10). Although we performed our study on a much smaller sample size, we detected titers of S1 or RDB-specific IgG of up to 1:3200. However, the majority of donors (54%) had titers below 1:200, and only a third of samples had high titers >1:800. Unsurprisingly, IgA and IgM titers were lower than IgG titers, and did not...

    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

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  3. Version published to 10.1101/2020.08.04.20167940v2 on medRxiv
  4. Version published to 10.1101/2020.08.04.20167940v1 on medRxiv