Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19
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SciScore for 10.1101/2021.11.10.468147: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animal use ethics statement: Hamsters were used according to protocols approved by the Institutional Animal Care and Use Committees of Colorado State University (CSU) Sex as a biological variable Animal experimental procedures: Male Syrian hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories and used at 12 weeks of age. Randomization not detected. Blinding The assays were performed blinded using the diluent as a negative control. Power Analysis The sample size was based on the number of available animals and resources of the collaborating investigator and not determined by a formal power calculation. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sent… SciScore for 10.1101/2021.11.10.468147: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animal use ethics statement: Hamsters were used according to protocols approved by the Institutional Animal Care and Use Committees of Colorado State University (CSU) Sex as a biological variable Animal experimental procedures: Male Syrian hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories and used at 12 weeks of age. Randomization not detected. Blinding The assays were performed blinded using the diluent as a negative control. Power Analysis The sample size was based on the number of available animals and resources of the collaborating investigator and not determined by a formal power calculation. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The assay plate was washed and 100 µL peroxidase conjugated goat anti-alpaca VHH domain-specific antibody (#128-035-232, Jackson ImmunoResearch), diluted to 0.8 µg/mL, transferred into each well and incubated for 1 hour at room temperature. anti-alpaca VHH domain-specific antibody (#128-035-232suggested: NoneExperimental Models: Cell Lines Sentences Resources Briefly, lentiviruses were propagated in HEK293T/17 cells (ATCC # CRL-11268) following published Current Protocols in Neuroscience.43 Plasmids for SARS-CoV-2 variants were synthesized using the prototype sequence (GenScript MC_0101081, human codon optimized, ER retention signal removed) as the reference sequence. HEK293T/17suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)Monolayer cultures of 293T cells were transiently transfected with plasmids expressing SARS-CoV-2 spike protein variants, psPAX2 (Addgene #12260), and a lentiviral transfer vector CD512-EF1a-RFP (System Biosciences CD512B-1) using Lipofectamine 2000. 293Tsuggested: NoneQuantification of SARS-CoV-2 in procedure samples: Virus titration was performed on oropharyngeal swabs obtained at 1, 2, and 3 days post infection and on cranial lung tissue samples obtained at 7 days post infection by double-overlay plaque assay on Vero E6 cells as previously described. Vero E6suggested: RRID:CVCL_XD71)Recombinant DNA Sentences Resources Monolayer cultures of 293T cells were transiently transfected with plasmids expressing SARS-CoV-2 spike protein variants, psPAX2 (Addgene #12260), and a lentiviral transfer vector CD512-EF1a-RFP (System Biosciences CD512B-1) using Lipofectamine 2000. psPAX2suggested: RRID:Addgene_12260)Software and Algorithms Sentences Resources X-ray diffraction data was collected at the SSRL beamline 9-2 and was processed with HKL3000.36 The structure was solved by molecular replacement in PHASER from the CCP4 suite37 using 7LDJ as a template in which the RBD moiety and nanobody were treated as independent searching models. CCP4suggested: (CCP4, RRID:SCR_007255)Iterative cycles of model building and refinement were done in Coot38 and Phenix. Phenixsuggested: (Phenix, RRID:SCR_014224)Structure validation and analysis: Ramachandran statistics were calculated with MolProbity40 and illustrations were prepared with PyMOL Molecular graphics (http://pymol.org). PyMOLsuggested: (PyMOL, RRID:SCR_000305)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Limitations of the study: While the current results provide strength for using nanobodies as potential inhaled therapeutics, there are several limitations to this current study. First, the use of bio-layer interferometry and pseudovirus assays to measure in vitro binding and neutralization provide evidence that NIH-CoVnb-112 has broad potency, yet these assays must be interpreted with caution. In the setting of authentic SARS-CoV-2 infection, and particularly the highly transmissible Delta variant, it is vital to assess the actual virus to ensure parity between in vitro and in vivo findings. The constellation of mutations in variants outside of the RBD may provide competitive fitness, which outstrips the ability of NIH-CoVnb-112 to effectively neutralize infection. Second, while acute weight loss and lung pathology provide metrics for assessing the early phase of infection, the natural course of disease in the Syrian hamster resolves with rare mortality.27 Thus, the hamster model should be considered just one of several relevant in vivo models. Third, the hamster experiments should be extended for future studies; the experiments presented here serve primarily as proof-of-concept for prophylactic and therapeutic potential. Given resource limitations, our studies had limitations on sample size and thus we have reported our findings without statistical analysis. We observed a robust viral load reduction and clear impact on lung pathology, which is encouraging but should not be c...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
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.
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