Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design
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Abstract
The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.
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SciScore for 10.1101/2021.05.15.444222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animal procedures were performed under the approvals of the Institutional Animal Care and Use Committee of University of Washington, Seattle, WA.
Euthanasia Agents: Mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 μL per injection site (100 μL total) of immunogen under isoflurane anesthesia.Sex as a biological variable Cell lines: Expi293F cells are derived from the HEK293F cell line, a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: … SciScore for 10.1101/2021.05.15.444222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animal procedures were performed under the approvals of the Institutional Animal Care and Use Committee of University of Washington, Seattle, WA.
Euthanasia Agents: Mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 μL per injection site (100 μL total) of immunogen under isoflurane anesthesia.Sex as a biological variable Cell lines: Expi293F cells are derived from the HEK293F cell line, a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies). Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cell lines other than Expi293F were not tested for mycoplasma contamination nor authenticated.
Authentication: Cell lines other than Expi293F were not tested for mycoplasma contamination nor authenticated.Table 2: Resources
Antibodies Sentences Resources Serial dilutions of sera were added to the plates and, after washing, antibody binding was revealed using a hydrogen peroxidase coupled horse anti-mouse IgG antibody. anti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines: Expi293F cells are derived from the HEK293F cell line, a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies). Expi293Fsuggested: RRID:CVCL_D615)HEK293Fsuggested: RRID:CVCL_6642)The HEK-ACE2 adherent cell line was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK293T-hACE2 Cell Line, NR-52511. HEK293Tsuggested: NoneBriefly, 293T-ACE2 cells (BEI NR-52511) were seeded at 1.25×104 cells per well in 50 uL D10 growth media (DMEM with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) in poly-L-lysine coated black-walled clear-bottom 96-well plates (Greiner 655930). 293T-ACE2suggested: RRID:CVCL_YZ65)Poly-lysine was removed, plates were dried for 5 min then washed 1× with water prior to plating cells HEK-hACE2 cells. HEK-hACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Mice: Female BALB/c mice four weeks old were obtained from Jackson Laboratory, Bar Harbor, Maine. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Recombinant DNA Sentences Resources The HexaPro-foldon construct used for immunization studies was produced as previously described (24) and placed into pCMV/R with an octa-histidine tag. pCMV/Rsuggested: NoneSoftware and Algorithms Sentences Resources Melting temperatures were defined as the maximum point of the first derivative of the melting curve, with first derivatives calculated using GraphPad Prism software after smoothing with four neighboring points using 2nd order polynomial settings. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Midpoint titers calculations (EC50) were generated based on fitted four point logistic equations using the SciPy library in Python, in which the EC50 was the serum dilution at which the curve reached 50% of its maximum. SciPysuggested: (SciPy, RRID:SCR_008058)Pythonsuggested: (IPython, RRID:SCR_001658)Results from OddPub: Thank you for sharing your code and data.
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.
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