A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

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   ScreenIT

   Apr 18, 2021

   SciScore for 10.1101/2021.04.16.440101: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Thirty-six female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 strain MUC-IMB-1 p.1 in a total volume of 50 μl.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   48-72 hours post-transfection, cells were harvested without trypsin treatment, washed in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) and used for binding assays or frozen in aliquots at −70°C. 105 thawed or freshly transfected cells were …

   SciScore for 10.1101/2021.04.16.440101: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Thirty-six female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 strain MUC-IMB-1 p.1 in a total volume of 50 μl.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   48-72 hours post-transfection, cells were harvested without trypsin treatment, washed in FACS buffer (PBS with 0.5% bovine serum albumin and 1 nmol sodium azide) and used for binding assays or frozen in aliquots at −70°C. 105 thawed or freshly transfected cells were incubated first in wells of a 96-well plate with 100μl undiluted hybridoma supernatant or 100 μl mouse serum (1:200 dilutions in R10+ medium), and bound antibodies were detected with a mix of Cy5-conjugated goat anti-pan-mouse IgG-Cy5 conjugated goat anti-mouse (Southern Biotechnology, Birmingham, USA, #SBA-1030-15) antibody.	anti-pan-mouse IgG-Cy5 suggested: None anti-mouse (Southern Biotechnology, suggested: None
   Briefly, 105 cells were fixed for 20 min in 2% paraformaldehyde (Morphisto, Frankfurt am Main, Germany) diluted in PBS, washed twice with FACS buffer, resuspended in permeabilization buffer (0.5% Saponin Sigma Aldrich, Taufkirchen, Germany, in FACS buffer) containing fluorochrome-conjugated murine H and L isotype-specific antibodies (antimouse IgG1-APC # 550874 and anti-mouse IgG3-bio # 020620 from BD, Franklin Lakes, USA, antimouse IgG2b-PE SBA-1090-09 and anti-mouse IgG2c-bio SBA-1079-08 from Southern Biotech, Birmingham, USA, anti-mouse Ig light chain lambda APC # 407306 and anti-mouse IgG light chain kappa PE both from Biolegend, San Diego, USA) and incubated for 1 hour at 4°C.	antimouse IgG1-APC suggested: None anti-mouse IgG3-bio suggested: None antimouse IgG2b-PE SBA-1090-09 suggested: None anti-mouse IgG2c-bio suggested: None anti-mouse Ig light chain lambda APC suggested: (BioLegend Cat# 407306, RRID:AB_961363) anti-mouse IgG suggested: None
   Competitional binding in the ELISA set up was achieved by applying 50 μl/well of 0.25 μg/ml human ACE2-Biotin (Acro Biosystems, Beijing, China, # A011-214) followed by 50 μl/well serially 2-fold pre-diluted TRES antibodies (2 μg/ml start concentration).	ACE2-Biotin suggested: None
   After 1 hour the cells were washed, and a goat anti-human IgG FITC (Jackson ImmunoResearch, West Grove, USA #109-096-088) antibody was applied.	anti-human IgG FITC suggested: (Jackson ImmunoResearch Labs Cat# 109-096-088, RRID:AB_2337666)
   The cells were incubated for 30min on ice, washed, and bound antibodies were detected with a mouse IgG2a Alexa647-conjugated antibody directed against human IgG-Fc (BioLegend, San Diego, USA #409320).	mouse IgG2a suggested: (BioLegend Cat# 409320, RRID:AB_2563330) human IgG-Fc suggested: None
   TRES6 and TRES328 antibodies were incubated for one hour at a concentration of 200 ng/ml with 2×106 TCID50 of the CoV2-ER1 virus.	TRES6 suggested: None TRES328 suggested: None
   SARS-CoV-2 infected cells were visualized using SARS-CoV-2 S protein specific immunochemistry staining with anti-SARS-CoV-2 spike glycoprotein S1 antibody (Abcam, Cambridge, Great Britain) as described previously [64].	anti-SARS-CoV-2 spike glycoprotein S1 suggested: (Abcam Cat# ab273074, RRID:AB_2847846)
   Experimental Models: Cell Lines
   Sentences	Resources
   Mice were boosted intramuscularly either with i) 5 μg of the S protein of SARS-CoV-2 stabilized in a pre-fusion conformation (designated SARS-CoV-2-S protein) and adjuvanted with 25 μg Monophosphoryl Lipid A (MPLA) liposomes (Polymun Scientific GmbH, Klosterneuburg, Austria) into the hind leg, ii) by electroporation of the DNA vaccines used for priming, or iii) with exosomes purified from HEK-293T cells transiently transfected with SARS-CoV-2 DNA as described previously [38].	HEK-293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Briefly, 2×108 spleen cells and inguinal lymph node cells (about 1×108 B cells) were mixed with 108 Sp2/0 cells and washed 3 times with RPMI1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) without FCS. 2 ml PEG was added dropwise within one minute to the suspended cell pellet.	Sp2/0 suggested: None
   The S protein was purified from transiently transfected HEK-293F cells as described previously [59].	HEK-293F suggested: RRID:CVCL_6642)
   Generation of escape mutants: 5×106 Vero-E6 cells were seeded on the day before infection in T175 flasks.	Vero-E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice: The TRIANNI C57/Bl6 mouse line HHKKLL was established in cooperation with TRIANNI (Patent US 2013/0219535 A1).	C57/Bl6 suggested: None
   Thirty-six female K18-hACE2 mice (Jackson Laboratory, Bar Harbor, USA) were infected intranasally under isoflurane anesthesia with 300 FFU of SARS-CoV-2 strain MUC-IMB-1 p.1 in a total volume of 50 μl.	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Software and Algorithms
   Sentences	Resources
   EC50 values were calculated by plotting hACE2 activity against antibody concentrations and applying a 4-parameter curve fit using GraphPad Prism 7.02 (San Diego, USA).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   The consensus sequences were analyzed using VDJsolver [60] and IMGT/V-Quest [61].	IMGT/V-Quest suggested: (IMGT/V-QUEST, RRID:SCR_010749)

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   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).

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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: We did not find any issues relating to colormaps.

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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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   Version published to 10.1101/2021.04.16.440101v1 on bioRxiv

   Apr 16, 2021