Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5

  1. Izumi Kimura
  2. Daichi Yamasoba
  3. Tomokazu Tamura
  4. Naganori Nao
  5. Tateki Suzuki
  6. Yoshitaka Oda
  7. Shuya Mitoma
  8. Jumpei Ito
  9. Hesham Nasser
  10. Jiri Zahradnik
  11. Keiya Uriu
  12. Shigeru Fujita
  13. Yusuke Kosugi
  14. Lei Wang
  15. Masumi Tsuda
  16. Mai Kishimoto
  17. Hayato Ito
  18. Rigel Suzuki
  19. Ryo Shimizu
  20. MST Monira Begum
  21. Kumiko Yoshimatsu
  22. Kanako Terakado Kimura
  23. Jiei Sasaki
  24. Kaori Sasaki-Tabata
  25. Yuki Yamamoto
  26. Tetsuharu Nagamoto
  27. Jun Kanamune
  28. Kouji Kobiyama
  29. Hiroyuki Asakura
  30. Mami Nagashima
  31. Kenji Sadamasu
  32. Kazuhisa Yoshimura
  33. Kotaro Shirakawa
  34. Akifumi Takaori-Kondo
  35. Jin Kuramochi
  36. Gideon Schreiber
  37. Ken J. Ishii
  38. Takao Hashiguchi
  39. Terumasa Ikeda
  40. Akatsuki Saito
  41. Takasuke Fukuhara
  42. Shinya Tanaka
  43. Keita Matsuno
  44. Kei Sato

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Abstract

No abstract available

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  1. Version published to 10.1016/j.cell.2022.09.018
  2. ScreenIT

    SciScore for 10.1101/2022.05.26.493539: (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 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)
    The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 minutes at 97°C to activate, and mouse anti-SARS-CoV-2 N monoclonal antibody (clone 1035111, R&D systems, Cat# MAB10474-SP, 1:400) was used as a primary antibody.
    anti-SARS-CoV-2 N
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T cells (a human embryonic kidney cell line; ATCC, CRL-3216)
    HEK293T
    suggested: None
    , HEK293 cells (a human embryonic kidney cell line; ATCC, CRL-1573) and HOS-ACE2/TMPRSS2 cells (HOS cells stably expressing human ACE2 and TMPRSS2) (Ferreira et al., 2021; Ozono et al., 2021) were maintained in DMEM (high glucose) (Sigma-Aldrich, Cat# 6429-500ML) containing 10% fetal bovine serum (FBS, Sigma-Aldrich Cat# 172012-500ML), and 1% penicillin-streptomycin (PS) (Sigma-Aldrich, Cat# P4333-100ML).
    HOS-ACE2/TMPRSS2
    suggested: None
    HEK293-ACE2/TMPRSS2 cells (HEK293 cells stably expressing human ACE2 and TMPRSS2) (Motozono et al., 2021) was maintained in DMEM (high glucose) containing 10% FBS, 1 µg/ml puromycin, 200 ng/ml hygromycin (Nacalai Tesque, Cat# 09287-84) and 1% PS.
    HEK293-ACE2/TMPRSS2
    suggested: None
    HEK293
    suggested: None
    HEK293-C34 cells (IFNAR1 KO HEK293 cells expressing human ACE2 and TMPRSS2 by doxycycline treatment) (Torii et al., 2021) were maintained in DMEM (high glucose) containing 10% FBS, 10 μg/ml blasticidin (InvivoGen, Cat# ant-bl-1) and 1% PS.
    HEK293-C34
    suggested: None
    Vero cells [an African green monkey (Chlorocebus sabaeus) kidney cell line; JCRB Cell Bank, JCRB0111] were maintained in Eagle’s minimum essential medium (EMEM) (Sigma-Aldrich, Cat# M4655-500ML) containing 10% FBS and 1% PS.
    Vero
    suggested: None
    VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2; JCRB Cell Bank, JCRB1819) (Matsuyama et al., 2020) 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)
    Calu-3 cells (a human lung epithelial cell line; ATCC, HTB-55) were maintained in EMEM (Sigma-Aldrich, Cat# M4655-500ML) containing 20% FBS and 1% PS.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Calu-3/DSP1-7 cells (Calu-3 cells stably expressing DSP1-7) (Yamamoto et al., 2020) were maintained in EMEM (Wako, Cat# 056-08385) containing 20% FBS and 1% PS. 293S GnTI(-) cells (HEK293S cells lacking N-acetylglucosaminyltransferase (Kubota et al., 2016) were maintained in DMEM (Nacalai tesque, #08458-16 containing 2% FBS without PS.
    HEK293S
    suggested: RRID:CVCL_A784)
    Briefly, the amount of pseudoviruses prepared was quantified by the HiBiT assay using Nano Glo HiBiT lytic detection system (Promega,Cat# N3040) as previously described (Ozono et al., 2021; Ozono et al., 2020), and the same amount of pseudoviruses (normalized to the HiBiT value, which indicates the amount of p24 HIV-1 antigen) was inoculated into HOS-ACE2/TMPRSS2 cells, HEK293-ACE2 cells or HEK293-ACE2/TMPRSS2 and viral infectivity was measured as described above (see “Neutralization assay” section).
    HEK293-ACE2
    suggested: None
    On day 3 (24 hours posttransfection), 16,000 effector cells were detached and reseeded into 96-well black plates (PerkinElmer, Cat# 6005225), and target cells (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
    SARS-CoV-2 infection: One day before infection, Vero cells (10,000 cells) and VeroE6/TMPRSS2 cells (10,000 cells) were seeded into a 96-well plate.
    VeroE6/TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Preparation of mouse sera: BALB/c mice (female, 7 weeks old) were immunized with 1 μg SARS-CoV-2 BA.2 RBD protein in 50% AddaVax (Invivogen, Cat# vac-adx-10) at day 0 and 14.
    BALB/c
    suggested: None
    Recombinant DNA
    SentencesResources
    The resulting PCR fragment was digested with KpnI and NotI and inserted into the corresponding site of the pCAGGS vector (Niwa et al., 1991).
    pCAGGS
    suggested: RRID:Addgene_127347)
    2 S RBD (residues 322-536) was cloned into the expression vector pHLsec containing the N-terminal secretion signal sequence and the C-terminal His6-tag sequence (Aricescu et al., 2006).
    pHLsec
    suggested: None
    kit (Roche, Cat# KK2601) and assembled in vivo by yeast [Saccharomyces cerevisiae strain EBY100 (ATCC, MYA-4941)] homologous recombination with pJYDC1 plasmid (Addgene, Cat# 162458) as previously described (Dejnirattisai et al., 2022; Kimura et al., 2022a; Kimura et al., 2022b; Motozono et al., 2021; Yamasoba et al., 2022a; Zahradnik et al., 2021a)
    pJYDC1
    suggested: RRID:Addgene_162458)
    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) (Kondo et al., 2011).
    pDSP1-7
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequencing reads were trimmed using fastp v0.21.0 (Chen et al., 2018) and subsequently mapped to the viral genome sequences of a lineage A isolate (strain WK-521; GISAID ID: EPI_ISL_408667) (Matsuyama et al., 2020) using BWA-MEM v0.7.17 (Li and Durbin, 2009).
    BWA-MEM
    suggested: (Sniffles, RRID:SCR_017619)
    Variant calling, filtering, and annotation were performed using SAMtools v1.9 (Li et al., 2009) and snpEff v5.0e (Cingolani et al., 2012).
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    The viral genome sequences were mapped to the reference sequence of Wuhan-Hu-1 (GenBank accession number: NC_045512.2) using Minimap2 v2.17 (Li, 2018) and subsequently converted to a multiple sequence alignment according to the GISAID phylogenetic analysis pipeline (https://github.com/roblanf/sarscov2phylo).
    Minimap2
    suggested: (Minimap2, RRID:SCR_018550)
    Tree reconstruction was performed by RAxML v8.2.12 (Stamatakis, 2014) under the GTRCAT substitution model.
    RAxML
    suggested: (RAxML, RRID:SCR_006086)
    Parameter estimation was performed via the MCMC approach implemented in CmdStan v2.28.1 (https://mc-stan.org) with CmdStanr v0.4.0 (https://mc-stan.org/cmdstanr/).
    CmdStan
    suggested: None
    https://mc-stan.org
    suggested: (Stan, RRID:SCR_018459)
    CmdStanr
    suggested: None
    The assay of each serum was performed in triplicate, and the 50% neutralization titer (NT50) was calculated using Prism 9 software v9.1.1 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    RBD expression and ACE2 signal were recorded by using a FACS S3e cell sorter device (Bio-Rad), background binding signals were subtracted and data were fitted to a standard noncooperative Hill equation by nonlinear least-squares regression using Python v3.7 (https://www.python.org) as previously described (Kimura et al., 2022a; Kimura et al., 2022b; Motozono et al., 2021; Yamasoba et al., 2022a; Zahradnik et al., 2021b).
    Python
    suggested: (IPython, RRID:SCR_001658)
    https://www.python.org
    suggested: (CVXOPT - Python Software for Convex Optimization, RRID:SCR_002918)
    Surface expression level of S proteins (Figures 3C and S2B) was measured using FACS Canto II (BD Biosciences) and the data were analyzed using FlowJo software v10.7.1 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The size of syncytium (GFP-positive area) was measured using Fiji software v2.2.0 (ImageJ) as previously described (Suzuki et al., 2022; Yamasoba et al., 2022a).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    The stained cells were washed with tap water and dried, and the size of plaques was measured using Fiji software v2.2.0 (ImageJ).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Images were incorporated as virtual slide by NDP.scan software v3.2.4 (Hamamatsu Photonics).
    NDP.scan
    suggested: None
    These analyses were performed in R 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2022.05.26.493539v1 on bioRxiv