Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters

  1. Neil C. Dalvie
  2. Sergio A. Rodriguez-Aponte
  3. Brittany L. Hartwell
  4. Lisa H. Tostanoski
  5. Andrew M. Biedermann
  6. Laura E. Crowell
  7. Kawaljit Kaur
  8. Ozan Kumru
  9. Lauren Carter
  10. Jingyou Yu
  11. Aiquan Chang
  12. Katherine McMahan
  13. Thomas Courant
  14. Celia Lebas
  15. Ashley A. Lemnios
  16. Kristen A. Rodrigues
  17. Murillo Silva
  18. Ryan S. Johnston
  19. Christopher A. Naranjo
  20. Mary Kate Tracey
  21. Joseph R. Brady
  22. Charles A. Whittaker
  23. Dongsoo Yun
  24. Swagata Kar
  25. Maciel Porto
  26. Megan Lok
  27. Hanne Andersen
  28. Mark G. Lewis
  29. Kerry R. Love
  30. Danielle L. Camp
  31. Judith Maxwell Silverman
  32. Harry Kleanthous
  33. Sangeeta B. Joshi
  34. David B. Volkin
  35. Patrice M. Dubois
  36. Nicolas Collin
  37. Neil P. King
  38. Dan H. Barouch
  39. Darrell J. Irvine
  40. J. Christopher Love

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Abstract

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). 1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. 2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs. 3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. 4–6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. 7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.03.433558: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All procedures were approved by the Massachusetts Institute of Technology Institutional Animal Care and Use Committee (IACUC) following local, state, and federal regulations.
    RandomizationImmunization of hamsters: Male and female Syrian golden hamsters (Envigo), 7-8 weeks of age, were randomly distributed into three groups.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableImmunization studies were carried out using age-matched 6-8 wk old Balb/cJ female mice purchased from The Jackson Laboratory (strain: 000651).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blood samples were collected on day 42 and sera tested for RBD specific antibodies in ELISA with the following modifications: Plates were coated with 1.25µg/mL soluble RBD (produced previously13) and mouse serum Ig was detected using a goat anti mouse Ig coupled to HRP (Southern Biotech) diluted at 1/6000.
    anti mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    , luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT were co-transfected into HEK293T cells by lipofectamine 2000 (ThermoFisher).
    HEK293T
    suggested: None
    To determine the neutralization activity of the plasma or serum samples from animals, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 104 cells/well overnight.
    HEK293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunization of mice for RBD adjuvant studies: The immunogenicity of RBD-L452K-F490W in combination with various adjuvants was evaluated in 7-8 week-old C57BL/6J female mice (Charles River, strain: 000634) at the Vaccine Formulation Institute (VFI, Switzerland).
    C57BL/6J
    suggested: None
    Immunization of mice for RBD-VLP studies: Balb/cJ female mice, ages 6-8 weeks were purchased from The Jackson Laboratory (strain: 000651).
    Balb/cJ
    suggested: RRID:IMSR_JAX:000651)
    Software and Algorithms
    SentencesResources
    Transcripts were quantified with Salmon version 1.1.04 and selective alignment using a target consisting of the K. phaffii transcripts, the RBD-N1del, P[8] and selectable marker transgene sequences and the K.
    Salmon
    suggested: (Salmon, RRID:SCR_017036)
    Expression values were summarized with tximport version 1.12.35 and edgeR version 3.26.8.6,7 Expression was visualized using log2(
    tximport
    suggested: (tximport, RRID:SCR_016752)
    edgeR
    suggested: (edgeR, RRID:SCR_012802)
    Gene set enrichment analysis (GSEA) was performed with GSEA 4.1.0 using Wald statistics calculated by DESeq28 and gene sets from yeast GO Slim.
    Gene set enrichment analysis
    suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    Data analysis was performed using the MicroCal LLC DSC plug-in for the Origin 7.0 software package.
    Origin
    suggested: (Origin, RRID:SCR_014212)
    Cells were lysed with a Microfluidics M110P microfluidizer, clarified by centrifugation, and captured from soluble lysate by IMAC, using Cytiva
    Cytiva
    suggested: (Image Quant TL, RRID:SCR_018374)
    RBD valency was determined using SDS-PAGE densitometry in ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    For each sample, ELISA endpoint titer was calculated in Graphpad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields an absorbance value (450nm) of 0.2.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    14–16 Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program)
    AIDS Resource and Reagent Program
    suggested: None

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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

    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 10.1101/2021.03.03.433558v1 on bioRxiv