A recombinant SARS-CoV-2 RBD antigen expressed in insect cells elicits immunogenicity and confirms safety in animal models

  1. Ricardo Choque-Guevara
  2. Astrid Poma-Acevedo
  3. Ricardo Montesinos-Millán
  4. Dora Rios-Matos
  5. Kristel Gutiérrez-Manchay
  6. Angela Montalvan
  7. Stefany Quiñones-Garcia
  8. Maria de Grecia Cauti-Mendoza
  9. Andres Agurto-Arteaga
  10. Ingrid Ramirez-Ortiz
  11. Manuel Criollo-Orozco
  12. Edison Huaccachi-Gonzales
  13. Yomara K. Romero Lázaro
  14. Norma Perez-Martinez
  15. Gisela Isasi-Rivas
  16. Yacory Sernaque-Aguilar
  17. Doris Villanueva-Pérez
  18. Katherine Vallejos-Sánchez
  19. Manolo Fernández-Sánchez
  20. Luis Guevara
  21. Manolo Fernández-Díaz
  22. Mirko Zimic
  23. for the COVID-19 Working Group in Perú

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Abstract

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus requires new immunization strategies in addition to the current vaccines approved for human administration. In the present report, the immunological and safety evaluation in mice and hamsters of a subunit vaccine based on the RBD sub-domain with two adjuvants of oil origin is described.

The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions.

In immunization assays in mice and hamsters, the purified RBD formulated with adjuvants based on oil-water emulsifications and squalene was able to stimulate specific neutralizing antibodies and confirm the secretion of IFN-γ after stimulating spleen cells with the purified RBD. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results demonstrate the potential of the purified RBD administered with adjuvants through an intramuscular route, to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection against infection.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.26.470043: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: The use of animals was aligned to ethical protocols approved by the Bioethics Committee of the Universidad Nacional Hermilio Valdizán and the animal’s ethical Committee at the Universidad Peruana Cayetano Heredia, registered as approval certificates of Research Project No. 1, 2, and 10 and E011-06-20, respectively
    Sex as a biological variableAnimals: This study used thirty-five female albino mice (Mus musculus) strain BALB/c of 5-8 weeks-old and 5 female Golden Syrian hamsters (Mesocricetus auratus) of 8-10 weeks-old obtained from the Universidad peruana Cayetano
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Protein fractions were collected and analyzed by SDS-PAGE under reducing conditions and Western blot using a commercial anti-His monoclonal antibody.
    anti-His
    suggested: None
    Five washing steps with PBS-T were performed, 100 µL of rabbit IgG polyclonal anti-spike antibody (SinoBiological, China) was added to the wells (1:5000) in 1% skimmed milk and incubated for 1 hour at 37ºC.
    anti-spike
    suggested: None
    After that, the mix was marked with rabbit monoclonal antibody anti-SARS-CoV-2 S1 (1:200) (Sino Biological, China) as the primary antibody for 1 h at 37°C, followed by the addition of the secondary goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:200) (Abcam, USA).
    anti-SARS-CoV-2 S1
    suggested: None
    the secondary goat
    suggested: None
    IgG antibody conjugated with Alexa Fluor 488 ( 1:200 ) ( Abcam , USA)
    suggested: None
    After three washes with TBS-T, anti-Hamster IgG antibody conjugated to HRP (Abcam, USA) was added to the membrane at 1:5000 dilution in 5% non-fat milk and incubated for two hours at room temperature.
    anti-Hamster IgG antibody
    suggested: None
    anti-Hamster IgG
    suggested: None
    Cells were fixed using the BD Cytofix/Cytoperm® kit (BD Biosciences, USA) following the manufacturer’s instructions, and then labeled with conjugated antibodies to surface antigens (PerCP-Cy®5.5 anti-mouse CD3, FITC anti-mouse CD4, APC-Cy®7 mouse anti-CD8, all from BD Biosciences, USA; LIVE/DEAD™ Fixable Yellow Dead Cell Stain, Invitrogen, USA) and intracellular cytokines (PE anti-mouse IFN-γ,
    anti-mouse CD4
    suggested: None
    anti-CD8
    suggested: None
    anti-mouse IFN-γ ,
    suggested: None
    Immunophenotype of spleen mononuclear cells: Mononuclear cells were directly labeled with conjugated antibodies to surface antigens (PerCP-Cy®5.5 anti-mouse CD3, clone, FITC anti-mouse CD4, APC-Cy®7 anti-mouse CD8, for T lymphocyte phenotype, all from BD Biosciences, USA; LIVE/DEAD™ Fixable Yellow Dead Cell Stain, for cell viability, cat. No. L34959, invitrogen, USA).
    anti-mouse CD3
    suggested: None
    anti-mouse CD8
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Propagation of baculovirus and expression of RBD in Sf9 insect cells culture: The recombinant baculovirus was amplified in Sf9 cells (Thermo Fisher Scientific, USA) to a density of 2 × 106 cells/mL in ExCell 420 medium (Sigma Aldrich, USA) supplemented with 5% fetal bovine serum (Gibco, USA).
    Sf9
    suggested: None
    RBD binding to Vero-E6 cells: Vero-E6 cells (Cod. CRL-1586™, ATCC®, USA), which were previously cultured in DMEM/F12 (HyClone, USA) + 10% fetal bovine serum (FBS) (HyClone, USA), were harvested and washed with DPBS with 5% FBS (FACS buffer).
    Vero-E6
    suggested: None
    To remove the excess of RBD not attached to Vero E6, the cells were washed with FACS buffer twice.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: This study used thirty-five female albino mice (Mus musculus) strain BALB/c of 5-8 weeks-old and 5 female Golden Syrian hamsters (Mesocricetus auratus) of 8-10 weeks-old obtained from the Universidad peruana Cayetano
    albino
    suggested: None
    Immunization and samples collection in mice: Female BALB/c mice (18-25g) were immunized intramuscularly (i.m.) with 20 and 50 µg/mice of purified RBD mixed with 50 µL of A1 or A3 (1:1, 100 µL final volume).
    BALB/c
    suggested: None
    Software and Algorithms
    SentencesResources
    10.6 (BD Biosciences, USA), and the graphics were generated with GraphPad Prism 8.0.1.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The labeled cells were acquired with the BD FACSCanto™ II flow cytometer and analyzed with the program FlowJo v.
    BD FACSCanto™
    suggested: None
    These cells were acquired with the BD FACSCanto™ II flow cytometer, and the analysis was performed with the program FlowJo v 10.6.2 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: All quantitative data were analyzed using GraphPad Prism version 6.1 (GraphPad Software, San Diego, CA, USA).
    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:
    Due to limitations in space and the availability of animals, this trial was conducted with 5 individuals per group, and the heterogeneity was evident as previously reported in a similar protocol [41]. Unfortunately, it was not possible to establish clear conclusions about the tendency of the population when stimulated with the two different adjuvants, as there was no significant difference between the controls and the immunized groups. It is important to perform additional studies with a greater sample size to perform a better evaluation of cellular and humoral immunity [13,16,23,28] to 8 per group [35,42]. In conclusion, the RBD vaccine candidate presented in this study, administered through an intramuscular route, was shown to be safe and able to induce humoral and cellular immunity as well as neutralizing antibodies in mice and hamsters. Further studies are required to evaluate protection in a challenge trial.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

    About SciScore

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  2. Version published to 10.1101/2021.11.26.470043v1 on bioRxiv