D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

  1. Drew Weissman
  2. Mohamad-Gabriel Alameh
  3. Thushan de Silva
  4. Paul Collini
  5. Hailey Hornsby
  6. Rebecca Brown
  7. Celia C. LaBranche
  8. Robert J. Edwards
  9. Laura Sutherland
  10. Sampa Santra
  11. Katayoun Mansouri
  12. Sophie Gobeil
  13. Charlene McDanal
  14. Norbert Pardi
  15. Nick Hengartner
  16. Paulo J.C. Lin
  17. Ying Tam
  18. Pamela A. Shaw
  19. Mark G. Lewis
  20. Carsten Boesler
  21. Uğur Şahin
  22. Priyamvada Acharya
  23. Barton F. Haynes
  24. Bette Korber
  25. David C. Montefiori

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.07.22.20159905: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Key Resources Table: Ethics statement: The clinical trial described in this manuscript was randomized double-blinded and the sera collections were approved by the appropriate IRBs.
    IACUC: All protocols, experimentation and animal manipulation adhered to the Guide for the Care and Use of Laboratory Animals by the Committee on Care of Laboratory Animal Resources Commission on Life Sciences, National Research Council under IACUC approved protocols. mRNA production: N1-methylpseudouridine modified mRNA was produced as previously described (Freyn et al., 2020) using T7 RNA polymerase (MegaScript, ThermoFisher Scientific, Waltham, MA, USA) on Not-I/AlfII double digested and linearized plasmids encoding codon-optimized di-proline modified pre-fusion SARS-CoV-2 Spike (Wuhan Hu-1 complete genome, GenBank: MN908947.3), full length S-furin mutant protein (RRAR furin cleavage site abolished), and RBD.
    Consent: Serum samples from people known to be infected with either the D614 or G614 form of SARS-CoV-2 were collected following informed consent from healthcare workers at Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK as part of the COVID-HERO SARS-CoV-2 seroprevalence study (Research Ethics Committee reference 20/HRA/2180).
    RandomizationKey Resources Table: Ethics statement: The clinical trial described in this manuscript was randomized double-blinded and the sera collections were approved by the appropriate IRBs.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Monoclonal antibodies: SARS-CoV-2 RBD-binding mAbs CR3022, B38, H4, P2B-2F6, and S309 were obtained from Dr. Peter Kwong at the Vaccine Research Center, National Institutes of Health, USA.
    H4
    suggested: None
    P2B-2F6
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293T/ACE2 cells were kindly provided by Drs.
    293T/ACE2
    suggested: RRID:CVCL_YZ65)
    Pseudovirions were produced in HEK 293T/17 cells (ATCC cat. no. CRL-11268) by transfection using Fugene 6 (Promega Cat#E2692) and a combination of Spike plasmid, lentiviral backbone plasmid (pCMV ΔR8.2) and firefly Luc reporter gene plasmid (pHR’ CMV Luc)(Naldini et al., 1996) in a 1:17:17 ratio.
    HEK 293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    Experimental Models: Organisms/Strains
    SentencesResources
    Administration of test articles (immunization) and blood collection: 8-12 week old Balb/c mice were immunized with either 10 or 30 µg of mRNA-LNP via the intramuscular (I.M.) or intradermal (I.D.) routes of administration using a 29G X1/2’’ Insulin syringe.
    Balb/c
    suggested: None
    Software and Algorithms
    SentencesResources
    Variants, including the A-to-G change at nucleotide 23,403 leading to D614G, were called using nanopolish (https://github.com/jts/nanopolish) and consensus sequences compared to the reference.
    https://github.com/jts/nanopolish
    suggested: (Nanopolish, RRID:SCR_016157)
    The RELION program (Scheres, 2016) was used for particle picking, 3D classification, and 3D refinements.
    RELION
    suggested: (RELION, RRID:SCR_016274)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04470427Active, not recruitingA Study to Evaluate Efficacy, Safety, and Immunogenicity of …
    NCT04368728Active, not recruitingStudy to Describe the Safety, Tolerability, Immunogenicity, …
    NCT04380701RecruitingA Trial Investigating the Safety and Effects of Four BNT162 …

    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.

    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 10.1016/j.chom.2020.11.012
  3. Version published to 10.1101/2020.07.22.20159905v2 on medRxiv
  4. NCRC

    Our take

    The D614G mutation in SARS-CoV-2 spike protein is unlikely to alter immunogenicity and derail current mRNA vaccines in development. Other vaccine platforms remain to be tested, but there is no evidence to suggest that their efficacy would be compromised by D614G. These results are altogether not surprising, as residue 614 in the spike proteiin is not part of the receptor-binding domain (RBD), and regions within the RBD are thought to be the primary immunodominant epitopes. Thus, relatively little concern was present a priori that this mutation would drastically alter SARS-CoV-2 immunogenicity.

    Study design

    other

    Study population and setting

    The mutation D614G in the spike glycoprotein (S) has garnered much attention and concern with SARS-CoV-2. Recent findings suggest this mutation may increase the in vitro infectivity of the virus and possibly the viral load in patients, although it is not thought to increase virulence. This study assessed the in vitro neutralization titers of sera from mice, non-human primates, and humans immunized with one of four different mRNA vaccines against lentiviral particles pseudotyped with full-length SARS-CoV-2 S bearing either the D614 or G614 polymorphism.

    Summary of main findings

    All four versions of the mRNA vaccine tested, which included the immunogens used in both the Moderna and BioNTech vaccines, which are currently in clinical trials, elicited similar in vitro neutralizing titers against both D614 and G614, with slightly higher neutralizing titers against the latter. Furthermore, sera from humans and macaques, which received mRNA vaccines encoding only for secreted RBD, also demonstrated this trend, suggesting that despite not being a structural component of the RBD, changes in residue 614 may elicit a conformational change in the RBD that simultaneously renders it both more capable of binding its ACE2 receptor and more susceptible to neutralizing antibodies.

    Study strengths

    Sera from both vaccinated animals and humans (from Phase I/II clinical trials) is utilized. The pseudotyped lentiviral particle system provides a very controlled environment in which to test only one variable: the D614G mutation.

    Limitations

    Only mRNA vaccine platforms were tested, as the authors note, and these constitute only a portion of the vaccine candidates currently in clinical and preclinical development. The in vitro studies performed with HEK293T/ACE2 cells and pseudotyped lentiviral particles, rather than SARS-CoV-2, are highly artificial, and the real-world applicability of the results is likely limited to some degree, as the authors acknowledge. Finally, the immunological correlates of protection against SARS-CoV-2 are yet to be definitively established, and although evidence suggests that neutralizing antibodies likely play an important role, other parameters (e.g. T-cell response) may prove equally important and are not evaluated here.

    Value added

    This is the first study to evaluate neutralizing titers generated from vaccination with mRNA platforms in the context of the prevalent D614G S mutation.

  5. ScreenIT

    SciScore for 10.1101/2020.07.22.20159905: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMethods Ethics statement All in vivo experiments described in this manuscript were randomized doubleblinded and approved by the University of Pennsylvania (UPenn) IACUC.RandomizationMethods Ethics statement All in vivo experiments described in this manuscript were randomized doubleblinded and approved by the University of Pennsylvania (UPenn) IACUC.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    293T/ACE2 cells were kindly provided by Drs.
    293T/ACE2
    suggested: None
    Pseudovirions were produced in HEK 293T/17 cells (ATCC cat. no. CRL-11268) by transfection using Fugene 6 (Promega Cat#E2692) and a combination of Spike plasmid lentiviral backbone plasmid (pCMV ∆R8.2) and firefly Luc reporter gene plasmid (pHR' CMV Luc)27 in a 1:17:17 ratio.
    HEK 293T/17
    suggested: ATCC Cat# CRL-11268, CVCL_1926
    Software and Algorithms
    SentencesResources
    This equalization of inter-protomer energetics results in a higher population of one-up Spike conformations, leading to increased encounter between RBD and ACE2 receptor and greater exposure of RBD domain for neutralization (Gnana, personal communication to be replaced with Biorxiv).
    Biorxiv
    suggested: (bioRxiv, SCR_003933)
    The RELION program28 was used for particle picking, 3D classification, and 3D refinements.
    RELION
    suggested: (RELION, SCR_016274)

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  6. Version published to 10.1101/2020.07.22.20159905v1 on medRxiv