Mosaic receptor-binding domain nanoparticles induce protective immunity against SARS-CoV-2 challenges

  1. Dan Bi Lee
  2. Hyojin Kim
  3. Ju Hwan Jeong
  4. Ui soon Jang
  5. You Yeon Chang
  6. Seokbeom Roh
  7. Hyunbum Jeon
  8. Eun Jeong Kim
  9. Su Yeon Han
  10. Jin Young Maeng
  11. Stefan Magez
  12. Magdalena Radwanska
  13. Ji Young Mun
  14. Hyun Sik Jun
  15. Gyudo Lee
  16. Min-Suk Song
  17. Hye-Ra Lee
  18. Mi Sook Chung
  19. Yun Hee Baek
  20. Kyung Hyun Kim

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Abstract

Recurrent spillovers of α- and β-coronaviruses (CoV) such as acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, SARS-CoV-2, and possibly human CoV (NL63, 229E, OC43, and HKU1) have caused serious morbidity and mortality worldwide. Six receptor binding domains (RBDs) derived from α- and β-CoV that are considered to have originated from animals and cross-infected humans were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric subunits, PCNA1, PCNA2, and PCNA3. These were used to form a scaffold-based mosaic multivalent antigen, 6RBD-np. Electron microscopic and atomic force microscopic images show a ring-shaped disk with six protruding RBDs, like jewels in a crown, with a size of 40 nm. Prime-boost immunizations with 6RBD-np in BALB/c mice elicited strong, dose-dependent antibody responses. In human angiotensin converting enzyme 2-transgenic mice, the same immunization induced full-protection against SARS-CoV-2 wild type and Delta challenges, resulting in a 100% survival rate. The mosaic 6RBD-np provides a potential platform for developing a pan-CoV vaccine against newly emerging SARS-CoV-2 variants and future CoV spillovers.

Significance

Despite the arsenal of COVID-19 vaccines, hospitalization and mortality associated with SARS-CoV-2 (acute respiratory syndrome coronavirus 2) variants remain high. There is an urgent need to develop next-generation COVID vaccines that provide broad protection against diseases by current and newly emerging SARS-CoV-2 variants. In this study, six receptor binding domains (RBDs) derived from α- and β-CoV were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric scaffolds. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens. The prime-boost immunization in BALB/c and human angiotensin converting enzyme 2-transgenic mice with the 6RBD-np elicited strong, dose-dependent antibody responses and induced full-protection against both the SARS-CoV-2 wild type (WT) and Delta challenges. This study provides proof-of-concept that the mosaic 6RBD-np induces 100% protection against SARS-CoV-2 WT and Delta. It provides the potential of co-displaying heterologous antigens for novel vaccine designs, which can be deployed countering future pandemics.

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    SciScore for 10.1101/2022.02.18.480994: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Animal procedures were performed under the approval of the Institutional Animal Care and Use Committee (DSUA-2021-006-008) of Duksung Women’s University (Seoul, Korea).
    Sex as a biological variableHuman embryonic kidney cells (HEK293T) is a female human embryonic kidney cell line (ATCC) and the HEK-ACE2 (derived from HEK293T cells) adherent cell line was obtained through BEI Resources, NIAID,
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Pathology and immunohistochemistry: For histopathological examination, mouse lung samples harvested at 5 dpi were fixed in 10% formalin neutralization buffer (Sigma Aldrich) and stained with H&E. Additionally, IHC staining was performed to detect the viral antigen with primary antibodies, rabbit SARS-CoV-2 nucleocapsid pAb (40589-T62, Sinobiological, Beijing, China), at a dilution of 1:10,000 and HRP-conjugated secondary antibodies, using the Ventana discovery ULTRA system (Roche, Indianapolis, IN, USA)
    SARS-CoV-2 nucleocapsid pAb ( 40589-T62 , Sinobiological , Beijing , China)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and animals: Expi293F cells derived from the 293F cell line (Life Technologies, Carlsbad, CA, USA) were grown in Expi293 expression medium (Life Technologies), cultured at 37°C with 8% CO2 and shaking at 150
    293F
    suggested: RRID:CVCL_D615)
    HEK293T expressing human angiotensin-converting enzyme 2 (hACE2), HEK293T-hACE2 cell line (NR-52511).
    HEK293T
    suggested: None
    HEK293T-hACE2
    suggested: RRID:CVCL_A7UK)
    HEK293T and HEK-ACE2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Texas, USA) supplemented with 10% fetal bovine serum (FBS) (Atlas Biologicals, Colorado, USA) and 1% penicillin-streptomycin (P/S) (Hyclone, Waltham, MA, USA).
    HEK-ACE2
    suggested: None
    Vero E6 cells were maintained in DMEM containing 10% FBS, 4.0 mM L-glutamine, 110 mg/L sodium pyruvate, 4.5 g/L D-glucose and 1% antibiotic-antimycotic at 37°C and 5% CO2.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Six-week old female B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA)
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Fusion proteins, SARS-CoV-2 RBD-SD1 WT-PCNA1-SARS-CoV RBD-SD1, MERS-CoV RBD-SD1-PCNA2-SARS-CoV-2 RBD-SD1 VAR, and hCoV 229E RBD-SD1-PCNA3-hCoV HKU1 RBD-SD1, were constructed with SGG linker sequences by overlapping PCR, which were termed S-PCNA1, M-PCNA2, and H-PCNA3, respectively.
    SARS-CoV-2 RBD-SD1 WT-PCNA1-SARS-CoV RBD-SD1 , MERS-CoV RBD-SD1-PCNA2-SARS-CoV-2 RBD-SD1 VAR
    suggested: None
    RBD-SD1-PCNA3-hCoV HKU1 RBD-SD1
    suggested: None
    Plasmids encoding each antigen were amplified in E. coli strain DH5α, which were prepared in high purity and concentration using the PureLink HiPure plasmid kit (Invitrogen).
    DH5α
    suggested: None
    Mouse immunizations: Female BALB/c mice aged five weeks old were purchased from Koatech (Pyeongtaek, Kyunggi-do, Korea).
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    To generate HEK293T cell line stably expressing hACE2, HEK293T cells were transfected with pCEP4-myc-hACE2 and cells were selected using 100 μg/mL of hygromycin B (Invitrogen, Logan, Utah, USA).
    pCEP4-myc-hACE2
    suggested: None
    For 6RBD-SD1 proteins, residues from 319 to 592 for SARS-CoV-2 WT and variant (VAR), 306 to 578 for SARS-CoV S, 367 to 657 for MERS-CoV S, 284 to 499 for hCoV 229E S, and 313 to 674 for hCoV HKU1 spike proteins as well as PCNA1, PCNA2, PCNA3_S170V were amplified from the pSecTag2A vector (Addgene) using the primers described in Extended Data Table 2.
    pSecTag2A
    suggested: RRID:Addgene_17839)
    Software and Algorithms
    SentencesResources
    All sequences were confirmed by automated sequencing (Macrogen).
    Macrogen
    suggested: (Macrogen, RRID:SCR_014454)
    The IC50 values were calculated with non-linear regression using GraphPad Prism 8 (GraphPad Software, Inc.
    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 10.1101/2022.02.18.480994v1 on bioRxiv