Neutralizing antibody-independent immunity to SARS-CoV-2 in hamsters and hACE-2 transgenic mice immunized with a RBD/Nucleocapsid fusion protein

  1. Julia T. Castro
  2. Marcílio J. Fumagalli
  3. Natalia S. Hojo-Souza
  4. Patrick Azevedo
  5. Natalia Salazar
  6. Bruna Rattis
  7. Simone G. Ramos
  8. Lídia Faustino
  9. Gregório G. Almeida
  10. Livia I. Oliveira
  11. Tomas G. Marçal
  12. Marconi Augusto
  13. Rubens Magalhães
  14. Bruno Cassaro
  15. Gabriela Burle
  16. Daniel Doro
  17. Jorge Kalil
  18. Edson Durigon
  19. Andrés Salazar
  20. Otávia Caballero
  21. Alexandre Machado
  22. João S. Silva
  23. Flávio da Fonseca
  24. Ana Paula Fernandes
  25. Santuza R. Teixeira
  26. Ricardo T. Gazzinelli

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Abstract

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.16.460663: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All patients were adults and were enrolled in the study after providing written informed consent.
    Sex as a biological variableAll individuals were between 18 and 70 years old (36±11, female:male ratio=3.2) (Extended Table I)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Detection of human, mouse and hamster antigen-specific antibodies: Plates were coated overnight with 0.4 μg/well of either N, RBD, S recombinant proteins, or alternatively 104 PFU/well of UV-inactivated SARS-CoV-2, and blocked for 2 hours with PBS containing 2% bovine serum albumin (PBS-2% BSA) at 37°C.
    SARS-CoV-2
    suggested: None
    Serum were serially diluted and the bronchoalveolar lavage (BALF) samples tested at 1:1 dilution in PBS-2% BSA and incubated for 1 hour at 37°C, and then incubated with anti-human IgG-HRP antibody (Fapon), anti-hamster IgG-HRP or anti-mouse total IgG, IgG1, IgG2c conjugated with streptavidin-HRP (Southern Biotech).
    anti-human IgG-HRP
    suggested: None
    anti-hamster IgG-HRP
    suggested: None
    anti-mouse total IgG
    suggested: None
    IgG1 , IgG2c
    suggested: None
    After 20 hours of culture, the plates were washed and incubated with biotinylated antibody anti-IFN-γ (XMG1.2 – BD).
    anti-IFN-γ
    suggested: None
    The secondary antibody Alexa Fluor 594 anti-mouse IgG (ThermoFisher) was added and the nucleus was stained with DAPI (ThermoFisher).
    The secondary antibody Alexa Fluor 594 anti-mouse IgG
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Immunofluorescence assays: A 16-well chamber slide (ThermoFisher) was coated with 104 Vero E6 cells/well and incubated overnight with SARS-CoV-2 in a multiplicity of infection (M.O.I) of 10.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice, hamsters, virus and ethics statement: Female C57BL/6 mice, 6-10 weeks old, were purchased from the Center for Laboratory Animal Facilities of the Federal University of Minas Gerais (CEBIO-UFMG)
    C57BL/6
    suggested: None
    Recombinant DNA
    SentencesResources
    Competent E. coli Star™ (DE3) were transformed with the pET24 vector with N or the RBD sequences and E. coli pRARE with the pET24_with SpiN.
    pET24
    suggested: RRID:Addgene_73142)
    pRARE
    suggested: RRID:Addgene_84650)
    Software and Algorithms
    SentencesResources
    The sequences were translated in silico using the standard genetic code and multiple aligned together with N or S proteins of Lineage B (Wuhan – EPI_ISL_402123) by the Kalign tool 46 with parameters 8.52 “gap extension penalty”, 54.90 “gap open penalty” and 4.42 “gap terminal penalty”.
    Kalign
    suggested: (Kalign, RRID:SCR_011810)
    Immunization, challenge and histopathology: Hamsters and C57BL/6 or hACE2 mice received two administrations at 21 days apart, containing 10 μg of RBD, N or SpiN adjuvanted with 50 μg of Hiltonol® (Poly ICLC, supplied by Oncovir, Washington, D.C.) 39,40, or with AddaVax™ (oil-in-water emulsion similar to MF59) in a volume of 1:1 AddaVax™:Antigen 50.
    AddaVax™
    suggested: None
    Images were processed with the software ImageJ version 2.1 for Mac.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Data were analyzed using FlowJo v10.5.3 software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Statistical analysis was conducted using GraphPad Prism 6.0 for Mac (GraphPad Inc, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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.

  2. Version published to 10.1101/2021.09.16.460663v1 on bioRxiv