Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery

Article activity feed

1. Version published to 10.1016/j.ymthe.2022.09.010

   Feb 1, 2023
2. 

   ScreenIT

   May 29, 2022

   SciScore for 10.1101/2022.05.27.493569: (What is this?)

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

   Table 1: Rigor

   Ethics

   Euthanasia Agents: Each serial dilution was then mixed 1:1 with 2,000 TCID50/mL SARS-CoV-2 variant virus (Delta variant: strain hCoV-19/USA/MD-HP05647/2021; Omicron variant: strain hCoV-19/USA/MD-HP20874/2021) and incubated for 1 h at + 37 °C ± 2 °C and 5% ± 0.5% of CO2. IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of San Raffaele Scientific Institute.

   Sex as a biological variable

   Female transgenic K18-hACE2 mice, aged 8-10 weeks, were infected via the intranasal route with 1×105 TCID50/mouse of SARS-Cov-2 variant Delta B.1.617.2 virus [hCoV-19/Italy/LOM-Milan-UNIMI9615/2021 (GISAID Accession ID: EPI_ISL_3073880)], obtained from the Laboratory of …

   SciScore for 10.1101/2022.05.27.493569: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: Each serial dilution was then mixed 1:1 with 2,000 TCID50/mL SARS-CoV-2 variant virus (Delta variant: strain hCoV-19/USA/MD-HP05647/2021; Omicron variant: strain hCoV-19/USA/MD-HP20874/2021) and incubated for 1 h at + 37 °C ± 2 °C and 5% ± 0.5% of CO2. IACUC: All experimental animal procedures were approved by the Institutional Animal Committee of San Raffaele Scientific Institute.
   Sex as a biological variable	Female transgenic K18-hACE2 mice, aged 8-10 weeks, were infected via the intranasal route with 1×105 TCID50/mouse of SARS-Cov-2 variant Delta B.1.617.2 virus [hCoV-19/Italy/LOM-Milan-UNIMI9615/2021 (GISAID Accession ID: EPI_ISL_3073880)], obtained from the Laboratory of Microbiology and Virology of San Raffaele Scientific Institute.
   Randomization	Images shown in all figures are representative of at least five random fields (scale bars are indicated).
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   One hour after infection, the virus solution was discarded and replaced by a volume of growth medium containing scFv76 or not-neutralizing scFv5 antibody, in a concentration ranging from 214 to 2.6 nM, in triplicate.	scFv5 suggested: None
   The modelling was performed with antigen-binding fragment (Fv) as antibody format.	antigen-binding fragment (Fv) suggested: None
   We analyzed 130 CoV-AbDab structures of antibodies (Abs) bound to RBDs (23) and found that 39 out of 42 entries with IGHV3-53/IGHV3-66 HCs, usually coupled with IGKV1-9 (16 Abs) or IGKV3-20 (10 Abs) LCs, share a common binding mode to the RBD.	IGKV3-20 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viral Neutralization in Calu-3 cells: To measure the SARS-CoV-2-neutralizing capability of scFv76, a live SARS-CoV-2 assay was performed by measuring the viral load in human lung adenocarcinoma Calu-3 cells, by real-time reverse transcription-quantitative PCR (RT-qPCR), 72 h after virus infection.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Thirty-five μL of each diluted sample/virus mix were then applied in octuplicate to Vero E6 cells seeded at a density of 104 cells/well in a 96-well plate at day -1.	Vero E6 suggested: None
   Cell-cell fusion assay: Human alveolar type II-like epithelial A549 cells and embryonic kidney 293T cells were obtained from ATCC (Manassas, VA).	293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   Cells were grown at 37 °C and 5% CO2, in RPMI-1640 (A549 cells) or DMEM (293T cells) medium (Euroclone) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics.	A549 suggested: None
   Generation of A549 cells stably expressing the human ACE2 receptor (A549-hACE2 cells) has been described previously (20).	A549-hACE2 suggested: RRID:CVCL_A5KB)
   Software and Algorithms
   Sentences	Resources
   Kinetic constants were obtained by BIAevaluation 3.2 software (	BIAevaluation suggested: (BIAevaluation Software, RRID:SCR_015936)
   Data were processed using GraphPad Prism software (V8.0) and the IC50 values calculated using a four-parameter logistic curve fitting approach.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Statistical analysis was performed using one-way ANOVA (Prism 6.0 software; GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Both models were independently refined with COOT (25) and PHENIX (26) using the full reconstruction and the local refined map, at 3.4 Å and 4.0 Å resolution, respectively.	COOT suggested: (Coot, RRID:SCR_014222)
   The images were prepared using ChimeraX (27) and Pymol (http://www.pymol.org/pymol).	Pymol suggested: (PyMOL, RRID:SCR_000305)
   Cryo-EM movies were deposited in the Electron Microscopy Public Image Archive under the accession code EMPIAR-10990.	Image Archive suggested: None

   ---

   Results from OddPub: Thank you for sharing your data.

   ---

   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   In the search of easily deployable therapeutic measures against COVID-19, we recently described 76clAbs, a cluster of human single chain antibody fragments that, in principle, could bypass all limitations of traditional monoclonal antibodies. Indeed, the use of monoclonal antibodies for the therapy of COVID-19 is being challenged by several issues: 1) difficulties in the deployment of therapy, being monoclonal antibodies parenteral drugs to be administered in hospital environments; 2) the risk of antibody-dependent enhancement (ADE) that can be ignited by different routes involving the immunoglobulin Fc interaction with Fc receptor (16) or with the ACE2, recently found to possibly act as a secondary receptor (17), or with Fcγ-expressing cells including monocytes and macrophages that, by triggering the inflammatory cell death, needed to abort the production of infectious virus, cause systemic inflammation that contributes to the severity of COVID-19 pathogenesis (18); 3) evasion properties of SARS-CoV-2 variants, particularly recently emerged Omicron lineages for which most of approved and investigational antibodies lost their neutralization activity (4-10). The single chain antibody format, because of its high stability, can be easily used for friendly self-administrable aerosol treatments. Furthermore, single-chain antibodies are, in principle, devoid of ADE risk because of lack of Fc sequence. 76clAbs, which were selected on the original SARS-CoV-2 Wuhan strain, were found ...

   ---

   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.

   ---

   Read the original source
3. 

   Version published to 10.1101/2022.05.27.493569 on bioRxiv

   May 27, 2022