Epidemic Preparedness—Leishmania tarentolae as an Easy-to-Handle Tool to Produce Antigens for Viral Diagnosis: Application to COVID-19

  1. Ilaria Varotto-Boccazzi
  2. Alessandro Manenti
  3. Francesca Dapporto
  4. Louise J. Gourlay
  5. Beatrice Bisaglia
  6. Paolo Gabrieli
  7. Federico Forneris
  8. Silvia Faravelli
  9. Valentina Bollati
  10. Diego Rubolini
  11. Gianvincenzo Zuccotti
  12. Emanuele Montomoli
  13. Sara Epis
  14. Claudio Bandi

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Abstract

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.

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  1. Version published to 10.3389/fmicb.2021.736530
  2. ScreenIT

    SciScore for 10.1101/2021.07.05.21260035: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: A collection of 80 human serum from asymptomatic subjects (UNICORN study; ethics committee of the University of Milan, approval number 17/20, approval date March 6, 2020) was tested at 1:100 dilution, while fifteen human sera (five high positive for SARS-CoV-2, five low positive for SARS-CoV-2 and five pre-pandemic negative for SARS-CoV-2) were tested starting from 1:100 to 1:25600 dilutions.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Following electrophoresis, proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories), according to standard protocols, before blocking for 5 min at room temperature with EveryBlot Blocking Buffer (Bio-Rad Laboratories) and incubation with a 1:3000 dilution of anti-6x HisTag-HRP antibody (Thermo Fisher) in the EveryBlot Blocking Buffer for 1 h.
    anti-6x HisTag-HRP
    suggested: None
    In particular, the proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) which were incubated for 5 min at room temperature with EveryBlot Blocking Buffer (Bio-Rad Laboratories) and then incubated with a 1:3000 dilution of anti-SARS/SARS-CoV-2 Coronavirus Spike Protein (subunit 1) Antibody (Thermo Fisher) in the blocking buffer for 1 h.
    anti-SARS/SARS-CoV-2 Coronavirus Spike Protein ( subunit 1
    suggested: None
    In-House Leishmania-RBD Enzyme-Linked Immunosorbent Assay (ELISA) IgG qualification: The IgG ELISA assay was qualified for the serological detection of SARS-CoV-2 specific antibodies in human serum samples using the RBD antigen (Lt-RBD) according to International Conference on Harmonization Guidelines on Validation of Analytical Procedures (Q2 (R1)) [36].
    Q2
    suggested: None
    Next, after the washing step, 100 μL/well of Goat anti-Human IgG-Fc Horse Radish Peroxidase (HRP)-conjugated antibody 1:100,000 (Bethyl Laboratories, Montgomery USA) were added.
    anti-Human IgG-Fc
    suggested: None
    Comparative evaluation of SARS-CoV-2 IgG antibodies using two different RBD antigens: Specific anti-SARS-CoV-2 IgG antibodies in human sera were detected by means of an in-house RBD assay using Lt-RBD produced in L. tarentolae cells and com-RBD, produced from HEK 293 cells and purchased from Sino Biological.
    SARS-CoV-2 IgG antibodies
    suggested: None
    anti-SARS-CoV-2 IgG
    suggested: None
    com-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To this aim, 1, 2, 3 and 4 µg/ml coating concentrations were tested against human positive and negative sera for SARS-CoV-2 and compared with 1 μg/mL Spike-RBD (commercially available from Sino Biological, China - hereafter com-RBD) expressed and purified from HEK 293 cells.
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Recombinant DNA
    SentencesResources
    The pLEXSY-sat2 vector integrates into the chromosomal 18S rRNA (ssu) locus of the L. tarentolae parasite.
    pLEXSY-sat2
    suggested: None
    Software and Algorithms
    SentencesResources
    After three washes with PBS + 0.1% (v/v) Tween-20 (PBS-T), the membrane was incubated for 5 min with the Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using the ChemiDoc Touch Imaging System (Bio-Rad
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Data were analyzed and plotted using the GraphPad Prism 7 (Graphpad Software)
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    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:
    However, the use of mammalian cells for the production of viral antigens presents potential limitations, in particular for rapid application and fast production in developing countries: highly specialized cell factories are indeed needed for efficient protein production in mammalian cells, and production costs are relatively high, compared to microbial-based systems. Our study underlines L. tarentolae as a system that is easy to manipulate and culture, providing a sound alternative for the rapid production of serodiagnostic proteins, even at the forefront of novel epidemics, e.g. in the presence of “spillover” events in tropical countries [42]. Accordingly, we emphasize that L. tarentolae can produce high protein yields that can easily be increased to industrial scale, by growing the parasites in bioreactors and harvesting proteins using high throughput strategies [25]. Consequently, this renders the Leishmania microfactory also potentially applicable in developing countries.

    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.

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

    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 10.1101/2021.07.05.21260035v1 on medRxiv