Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike

  1. Daichi Yamasoba
  2. Izumi Kimura
  3. Hesham Nasser
  4. Yuhei Morioka
  5. Naganori Nao
  6. Jumpei Ito
  7. Keiya Uriu
  8. Masumi Tsuda
  9. Jiri Zahradnik
  10. Kotaro Shirakawa
  11. Rigel Suzuki
  12. Mai Kishimoto
  13. Yusuke Kosugi
  14. Kouji Kobiyama
  15. Teppei Hara
  16. Mako Toyoda
  17. Yuri L. Tanaka
  18. Erika P. Butlertanaka
  19. Ryo Shimizu
  20. Hayato Ito
  21. Lei Wang
  22. Yoshitaka Oda
  23. Yasuko Orba
  24. Michihito Sasaki
  25. Kayoko Nagata
  26. Kumiko Yoshimatsu
  27. Hiroyuki Asakura
  28. Mami Nagashima
  29. Kenji Sadamasu
  30. Kazuhisa Yoshimura
  31. Jin Kuramochi
  32. Motoaki Seki
  33. Ryoji Fujiki
  34. Atsushi Kaneda
  35. Tadanaga Shimada
  36. Taka-aki Nakada
  37. Seiichiro Sakao
  38. Takuji Suzuki
  39. Takamasa Ueno
  40. Akifumi Takaori-Kondo
  41. Ken J. Ishii
  42. Gideon Schreiber
  43. Hirofumi Sawa
  44. Akatsuki Saito
  45. Takashi Irie
  46. Shinya Tanaka
  47. Keita Matsuno
  48. Takasuke Fukuhara
  49. Terumasa Ikeda
  50. Kei Sato

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Abstract

No abstract available

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  1. Version published to 10.1016/j.cell.2022.04.035
  2. ScreenIT

    SciScore for 10.1101/2022.02.14.480335: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    To construct the plasmids expressing anti-SARS-CoV-2 monoclonal antibodies (Casirivimab, Imdevimab or Sotrovimab), the sequences of the variable regions of these antibodies were obtained from KEGG Drug Database (https://www.genome.jp/kegg/drug/) and were artificially synthesized (Fasmac).
    anti-SARS-CoV-2
    suggested: None
    To measure the surface expression level of S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-112048, 1:100).
    anti-SARS-CoV-2 S
    suggested: None
    Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) was used as negative controls, and APC-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 111-136-144, 1:50) was used as a secondary antibody.
    anti-rabbit IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 111-136-144, RRID:AB_2337987)
    6c), HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were stained with rabbit anti-TMPRSS2 polyclonal antibody (BIOSS, Cat# BS-6285R, 1:100).
    anti-TMPRSS2
    suggested: (Bioss Cat# bs-6285R, RRID:AB_11102333)
    For protein detection, the following antibodies were used: mouse anti-SARS-CoV-2 S monoclonal antibody (clone 1A9, GeneTex, Cat# GTX632604, 1:10,000), rabbit anti-beta actin (ACTB) monoclonal antibody (clone 13E5, Cell Signalling, Cat# 4970, 1:5,000), horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 711-035-152, 1:10,000) and HRP-conjugated donkey anti-mouse IgG polyclonal antibody (Jackson ImmunoResearch, Cat# 715-035-150, 1:10,000).
    anti-beta actin
    suggested: None
    ACTB
    suggested: (Cell Signaling Technology Cat# 4970, RRID:AB_2223172)
    HRP)-conjugated donkey anti-rabbit IgG
    suggested: None
    HRP-conjugated donkey anti-mouse IgG
    suggested: None
    The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 m at 97°C to activate, and mouse anti-SARS-CoV-2 N monoclonal antibody (R&D systems, Clone 1035111, Cat# MAB10474-SP, 1:400) was used as a primary antibody.
    anti-SARS-CoV-2 N
    suggested: (Leinco Technologies Cat# LT7000, RRID:AB_2893978)
    Experimental Models: Cell Lines
    SentencesResources
    HEK293-ACE2/TMPRSS2 cells [HEK293 cells (ATCC CRL-1573) stably expressing human ACE2 and TMPRSS2]22 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (Wako, Cat# 044-29765) containing 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (PS).
    HEK293-ACE2/TMPRSS2
    suggested: None
    VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2; JCRB1819)29 were maintained in DMEM (low glucose) (Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Nacalai Tesque, Cat# G8168-10ML) and 1% PS.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    JCRB1819)29
    suggested: None
    Calu-3/DSP1-7 cells [Calu-3 cells (ATCC HTB-55) stably expressing DSP1-7]40 were maintained in EMEM (Wako, Cat# 056-08385) supplemented with 20% FBS and 1% PS.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Expi293 cells (Thermo Fisher Scientific, Cat# A14527) were maintained in Expi293 expression medium (Thermo Fisher Scientific, Cat# A1435101).
    Expi293
    suggested: RRID:CVCL_D615)
    Briefly, the pCAGGS vectors containing the sequences encoding the immunoglobulin heavy and light chains were cotransfected into HEK293T cells using PEI Max (Polysciences, Cat# 24765-1).
    HEK293T
    suggested: None
    For the immunisation, mice were subcutaneously immunized with the freeze-thawed S-expressing B16F10 cells in complete Freund’s adjuvant (50%) (Sigma-Aldrich, Cat# F5881).
    B16F10
    suggested: None
    To produce chimeric recombinant SARS-CoV-2, the CPER products were transfected into HEK293-C34 cells using TransIT-LT1 (Takara, Cat# MIR2300) according to the manufacturer’s protocol.
    HEK293-C34
    suggested: None
    SARS-CoV-2 infection: One day before infection, Vero cells (10,000 cells), VeroE6/TMPRSS2 cells (10,000 cells), Calu-3 cells (20,000 cells), HEK293-ACE2 cells (10,000 cells), HEK293-ACE2/TMPRSS2 cells (10,000 cells), were seeded into a 96-well plate.
    Vero
    suggested: None
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Target HEK293 cells in selected wells were cotransfected with pC-TMPRSS2 (40 ng) in addition to the plasmids above.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    VeroE6/TMPRSS2 cells, Calu-3 cells, HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells were transfected with pDSP1-7 (400ng).
    HEK293-ACE2
    suggested: None
    On day 3 (24 h posttransfection), 16,000 effector cells were detached and reseeded into 96-well black plates (PerkinElmer, Cat# 6005225), and target cells (HEK293, VeroE6/TMPRSS2 or Calu-3/DSP1-7 cells) were reseeded at a density of 1,000,000 cells/2 ml/well in 6-well plates.
    Calu-3/DSP1-7
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    BALB/c mice (female, 7 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan).
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Recombinant DNA
    SentencesResources
    The obtained coding sequences of the variable regions of the heavy and light chains were cloned into the pCAGGS vector containing the sequences of the human immunoglobulin 1 and kappa constant region [kindly provided by Dr. Hisashi Arase (Osaka University, Japan)].
    pCAGGS
    suggested: RRID:Addgene_127347)
    To prepare effector cells, HEK293 cells were cotransfected with the S-expression plasmids (500 ng) and pDSP8-11 (500 ng) using PEI Max (Polysciences, Cat# 24765-1).
    pDSP8-11
    suggested: None
    To prepare target cells, HEK293 and HEK293-ACE2/TMPRSS2 cells were transfected with pDSP1-7 (500 ng).
    pDSP1-7
    suggested: None
    To prepare target cells, HEK293 cells were cotransfected with pC-ACE2 (200 ng) and pDSP1-7 (400 ng).
    pC-ACE2
    suggested: None
    Target HEK293 cells in selected wells were cotransfected with pC-TMPRSS2 (40 ng) in addition to the plasmids above.
    pC-TMPRSS2
    suggested: None
    An enhanced yeast display platform for SARS-CoV-2 RBD [wild-type (B.1.1), residues 336-528] yeast surface expression was established using Saccharomyces cerevisiae EBY100 strain and pJYDC1 plasmid (Addgene, Cat# 162458) as previously described22, 24, 47.
    pJYDC1
    suggested: RRID:Addgene_162458)
    Software and Algorithms
    SentencesResources
    Paired-end, 76-bp sequencing was performed using MiSeq (Illumina) with MiSeq reagent kit v3 (Illumina, Cat# MS-102-3001).
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Sequencing reads were trimmed using fastp v0.21.028 and subsequently mapped to the viral genome sequences of a lineage A isolate (strain WK-521; GISAID ID: EPI_ISL_408667)29 using BWA-MEM v0.7.1730.
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    Variant calling, filtering, and annotation were performed using SAMtools v1.931 and snpEff v5.0e32.
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    snpEff
    suggested: (SnpEff, RRID:SCR_005191)
    The alignment sites with >50% sequences having a gap or undetermined/ambiguous nucleotide were trimmed using trimAl v1.234.
    trimAl
    suggested: (trimAl, RRID:SCR_017334)
    The tree reconstruction was performed by RAxML v8.2.1235 under the GTRCAT substitution model.
    RAxML
    suggested: (RAxML, RRID:SCR_006086)
    A time-calibrated tree of each lineage was constructed by BEAST2 v.2.6.636.
    BEAST2
    suggested: (BEAST2, RRID:SCR_017307)
    Parameter estimation was performed by MCMC implemented in CmdStan v2.28.1 (https://mc-stan.org) with cmdstanr v0.4.0 (https://mc-stan.org/cmdstanr/).
    CmdStan
    suggested: None
    To construct the plasmids expressing anti-SARS-CoV-2 monoclonal antibodies (Casirivimab, Imdevimab or Sotrovimab), the sequences of the variable regions of these antibodies were obtained from KEGG Drug Database (https://www.genome.jp/kegg/drug/) and were artificially synthesized (Fasmac).
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Nucleotide sequences were determined by DNA sequencing services (Eurofins), and the sequence data were analyzed by Sequencher v5.1 software (Gene Codes Corporation).
    Sequencher
    suggested: (Sequencher, RRID:SCR_001528)
    The assay of each serum was performed in triplicate, and the 50% neutralisation titre (NT50) was calculated using Prism 9 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Information on the unexpected mutations detected is summarized in Supplementary Table 6, and the raw data are deposited in Gene Expression Omnibus (accession number: GSE196649).
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    The background binding subtracted fluorescent signal was fitted to a standard noncooperative Hill equation by nonlinear least-squares regression using Python v3.7 (https://www.python.org) as previously described47.
    Python
    suggested: (IPython, RRID:SCR_001658)
    https://www.python.org
    suggested: (CVXOPT - Python Software for Convex Optimization, RRID:SCR_002918)
    Surface expression level of TMPRSS2 was measured using FACS Canto II (BD Biosciences) and the data were analysed using FlowJo software v10.7.1 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Bands were visualized using an Amersham Imager 600 (GE Healthcare), and the band intensity was quantified using Image Studio Lite v5.2 (LI-COR Biosciences) or Fiji software v2.2.0 (ImageJ).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The state of oxygenation was examined by measuring SpO2 using pulse oximeter, MouseOx PLUS (STARR).
    STARR
    suggested: (Starr, RRID:SCR_001071)
    These analyses were performed in v4.1.2 (https://www.r-project.org/).
    https://www.r-project.org/
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)

    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.

    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.

  3. Version published to 10.1101/2022.02.14.480335v1 on bioRxiv