An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

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   ScreenIT

   Feb 20, 2022

   SciScore for 10.1101/2022.02.17.480751: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Animal protection studies were carried out under the animal permit PROEX-146.6/20, approved by the Community of Madrid (Spain), and performed in biosafety level 3 facilities at CISA-INIA (Madrid). Euthanasia Agents: Antibody injection, with challenge virus and euthanasia were performed under isoflurane anesthesia.
   Sex as a biological variable	Both female and male H2L2 mice were used.
   Randomization	not detected.
   Blinding	During the evaluation, the pathologist was blinded regarding the treatment groups and used virus strains.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Anti…

   SciScore for 10.1101/2022.02.17.480751: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Animal protection studies were carried out under the animal permit PROEX-146.6/20, approved by the Community of Madrid (Spain), and performed in biosafety level 3 facilities at CISA-INIA (Madrid). Euthanasia Agents: Antibody injection, with challenge virus and euthanasia were performed under isoflurane anesthesia.
   Sex as a biological variable	Both female and male H2L2 mice were used.
   Randomization	not detected.
   Blinding	During the evaluation, the pathologist was blinded regarding the treatment groups and used virus strains.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cell lines tested negative for mycoplasma.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Antigen specific antibody titres were monitored during immunization by taking blood samples from the mice and performing antigen-specific ELISA.	antigen-specific ELISA . suggested: None
   Gene blocks encoding the variable heavy (VH) and light (VL) chain sequences of 87G7 and of benchmark monoclonal antibodies REGN10933, REGN10987 (PDB ID: 6XDG) (43), S309 (PDB ID: 6WPS) (44), CR3022 (GenBank accession numbers: DQ168569.1 and DQ168570.1) (45), 47D11 (GenBank accession numbers: MW881223.1 and MW881224.1) (41) were synthesized.	CR3022 suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
   After 8 h of infection, the cells were fixed with formalin, permeabilized with 70% ethanol, washed in PBS and stained using rabbit anti-SARS-CoV nucleocapsid (SinoBiological, 1:2000 in 0.1% bovine serum albumin (BSA) in PBS) followed by goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen, 1:2000 in 0.1% BSA in PBS).	anti-SARS-CoV nucleocapsid suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503) anti-rabbit suggested: None
   87G7 or a non-SARS-CoV-2 human IgG control antibody were injected intraperitoneally in a volume of 500 µl.	human IgG suggested: None
   In this case, after 5 day incubation, cells were fixed with 4% PFA and stained using an anti-SARS-CoV-2 nucleocapsid antibody (Sinobiological).	anti-SARS-CoV-2 nucleocapsid antibody (Sinobiological). suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Recombinant human antibodies were expressed in HEK-293T cells following transient transfection with pairs of the IgG1 heavy and light chain expression plasmids.	HEK-293T suggested: None
   In the virus neutralization assay, 3-fold serially diluted mAbs were pre-incubated with an equal volume of virus at RT for 1 h, and then inoculated on VeroE6 cells, and further incubated at 37°C.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Omicron samples were titrated in Calu-3 cells due to the low infectivity of Omicron in Vero cells.	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609) Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse challenge experiment: In vivo prophylactic and therapeutic efficacy of mAb 87G7 against challenge with SARS-CoV-2 and four variants of concern, was evaluated in heterozygous K18-hACE2 C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) obtained from The Jackson Laboratory.	C57BL/6J suggested: RRID:MGI:3589388) 2B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: None
   Recombinant DNA
   Sentences	Resources
   All proteins were expressed transiently in HEK-293T (ATCC® CRL-11268™) cells from pCAGGS expression plasmids, and secreted proteins were purified from culture supernatants using streptactin beads (IBA) following the manufacturer’s protocol.	pCAGGS suggested: RRID:Addgene_127347)
   VH and VL sequences were separately cloned into the expression plasmids with human IgG1 heavy chain and human kappa chain constant regions, respectively using the HBM vectors pHBM 000254 (VH into pTT5-mIGK-hIgG1_HCv2) and HBM 000265 (VK into pTT5mIgK-hIgG_KCv2)	pTT5-mIGK-hIgG1_HCv2 suggested: None pTT5mIgK-hIgG_KCv2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Data were analyzed using ImageQuantTL 8.2 image analysis software (GE Healthcare).	ImageQuantTL suggested: None
   The PRNT titer was calculated using Graphpad Prism 9, calculating a 50% reduction in infected cells counts based on non-linear regression with bottom constraints of 0% and top constraints of 100%.	Graphpad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Particles were imported into Relion 3.1 and, using the “relion_particle_symmetry_expand” tool, each particle from the C3-symmetry–imposed reconstruction was assigned three orientations corresponding to its symmetry related views.	Relion suggested: (RELION, RRID:SCR_016274)
   Iterative rounds of manual fitting in Coot and real space refinement in Phenix (57) were carried out to improve non-ideal rotamers, bond angles and Ramachandran outliers.	Coot suggested: (Coot, RRID:SCR_014222)
   During refinement with Phenix, secondary structure and non-crystallographic symmetry restraints were imposed.	Phenix suggested: (Phenix, RRID:SCR_014224)
   The final model was validated with MolProbity (58), EMRinger (59) and Privateer (glycans) (60, 61).	MolProbity suggested: (MolProbity, RRID:SCR_014226)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 35. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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

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   Version published to 10.1101/2022.02.17.480751v1 on bioRxiv

   Feb 17, 2022