Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative

  1. Sameer Kumar Malladi
  2. Unnatiben Rajeshbhai Patel
  3. Raju S. Rajmani
  4. Randhir Singh
  5. Suman Pandey
  6. Sahil Kumar
  7. Sara Khaleeq
  8. Petrus Jansen van Vuren
  9. Shane Riddell
  10. Sarah Goldie
  11. Savitha Gayathri
  12. Debajyoti Chakraborty
  13. Parismita Kalita
  14. Ishika Pramanick
  15. Nupur Agarwal
  16. Poorvi Reddy
  17. Nidhi Girish
  18. Aditya Upadhyaya
  19. Mohammad Suhail Khan
  20. Kawkab Kanjo
  21. Madhuraj Bhat
  22. Shailendra Mani
  23. Sankar Bhattacharyya
  24. Samreen Siddiqui
  25. Akansha Tyagi
  26. Sujeet Jha
  27. Rajesh Pandey
  28. Shashank Tripathi
  29. Somnath Dutta
  30. Alexander J. McAuley
  31. Nagendrakumar Balasubramanian Singanallur
  32. Seshadri S. Vasan
  33. Rajesh P. Ringe
  34. Raghavan Varadarajan

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Abstract

No abstract available

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  1. Version published to 10.1021/acsinfecdis.1c00276
  2. Version published to 10.1101/2021.01.13.426626v3 on bioRxiv
  3. Version published to 10.1101/2021.01.13.426626v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2021.01.13.426626: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal studies were approved by the Institutional Animal Ethics committee (IAEC) (RR/IAEC/61-2019, Invivo/GP/084).
    IRB: The ethics approval of human clinical samples were approved by Institute Human Ethical Committee (Approval No: CSIR-IGIB/IHEC/2020-21/01) CPE based viral Neutralization assay: Mice and Guinea pig pre-immune (negative control) sera and post boost sera were assayed for virus neutralization.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMice and Guinea Pig Immunizations: Immunizations of BALBc mice (n=5/group, female, 3-4 weeks old, ∼16-18 g) and Hartley strain guinea pigs (n=5/group, female, 6-8 weeks old, ∼300 g) were performed with freshly adjuvanted (AddaVax™ (vac-adx-10)) protein (1:1 v/v Antigen : AddaVax™ ratio per animal/dose, 20 µg protein in 50 µl PBS (pH 7.4) and 50 µl AddaVax™) (InvivoGen, USA).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Trimeric RBD, ACE2-hFc and antibody expression constructs: The present trimeric mRBD construct consists of an N-terminal trimerization domain of human cartilage matrix protein (hCMP) (hCMP residues 298-340) (accession number AAA63904) linked by a 14-residue flexible linker (ASSEGTMMRGELKN) derived from the V1 loop of HIV-1 JR-FL gp120 linked to RBD residues 332-532 (accession number YP_009724390.1) with an engineered glycosylation site (NGS) at N532 fused to an HRV-3C precision protease cleavage site linked to a 10x Histidine tag by a GS linker.
    gp120
    suggested: (Fitzgerald Industries International Cat# 10R-G114a, RRID:AB_1285924)
    The expression levels were checked using a dot blot analysis with Anti-his tag antibodies conjugated with HRP enzyme.
    Anti-his tag
    suggested: None
    SPR-binding of hCMP-mRBD analyte to immobilized ACE2-hFc/CR3022: hCMP-mRBD protein kinetic binding studies to ACE2-hFc and CR3022 antibody were performed on a ProteOn XPR36 Protein Interaction Array V.3.1 (Bio-Rad).
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    Next, three washes were performed (200 µL of PBST/well) and anti-Human IgG secondary antibody (Sigma-Aldrich)
    anti-Human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Generation of adherent polyclonal Flp-In stable lines: T25 flasks (5 ml media) having either adherent Flp-In™-293 or Flp-In™-CHO cells (∼80 % confluent) were co-transfected with pOG44 (10 µg) and hCMP-mRBD-HRV-Tg-pcDNA5/FRT/TO (5µg) plasmid DNA using 35 µg of Lipofectamine™ 2000 Transfection Reagent (Thermo Fisher Scientific, Cat # 11668030) in serum free media as per the manufacturers instruction for 4 hr.
    Flp-In
    suggested: RRID:CVCL_U423)
    (Thermofisher Scientific Cat# 10687010) for Flp-In™-293 and 750 µg/ ml for Flp-In™-CHO cells.
    Flp-In™-293
    suggested: RRID:CVCL_U421)
    Tagless protein purification: The spent media from stable hCMP-mRBD-HRV-Tg-Flp-In™-293 or Flp-In™-CHO grown cells contained the expressed protein.
    Flp-In™-CHO
    suggested: RRID:CVCL_U424)
    The virus-serum incubated premix samples were serially diluted and plated in duplicates in VeroE6 cell containing 96 well plate (104/well) and cultured for 48/96 hours.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Briefly, HEK293T cells were transiently transfected with plasmid DNA pHIV-1 NL4.3Δenv-Luc and Spike-Δ19-D614G by using Profection mammalian transfection kit (Promega Inc) following the instructions in the kit manual.
    HEK293T
    suggested: None
    Patient derived convalescent sera (n = 40) were tested for neutralization in both 293T-ACE-2 and Vero/TMPRSS2 cells whereas animal sera were tested only in Vero/TMPRSS2 cells.
    Vero/TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    The correlation coefficients for ACE2-hFc receptor competition, pseudovirus, live virus and 293T-ACE2/VeroE6-TMPRSS2 cell line pseudovirus neutralizations were analysed by Spearman correlation using the GraphPad Prism software.
    293T-ACE2/VeroE6-TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    The unbound tagless proteins concentration was determined by absorbance (A280) using NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy)
    ProtParam
    suggested: (ProtParam Tool, RRID:SCR_018087)
    ExPASy
    suggested: None
    Reference-free 2D classification using single-particle analysis: The evaluation of micrographs was done with EMAN 2.1 (58).
    EMAN
    suggested: (EMAN, RRID:SCR_016867)
    Reference free 2D classification of different projections of particle were calculated using simple_prime2D of SIMPLE 2.1 software (59).
    SIMPLE
    suggested: (SIMPLE, RRID:SCR_009389)
    Statistical Analysis: The p values for ELISA binding titers, neutralization titers, ACE2 receptor competition titers were analysed with a two-tailed Mann-Whitney test using the GraphPad Prism software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

  5. Version published to 10.1101/2021.01.13.426626v1 on bioRxiv