Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

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1. Version published to 10.1093/pnasnexus/pgac045

   Apr 14, 2022
2. 

   ScreenIT

   Jan 3, 2022

   SciScore for 10.1101/2021.12.29.21268487: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Patients are part of the COVID-19 Registry of the LMU Klinikum (CORKUM, WHO trial id DRKS00021225) and the study was approved on March 23, 2020 by the ethics committee (no. 20-245) of the Faculty of Medicine of the LMU (Ethik-Kommission
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Membranes were incubated at 7°C overnight with primary anti-spike antibody (43A11, rat IgG, Helmholtz Zentrum Mu□nchen; 1A9, mouse IgG, GeneTex; PA5-81795, polyclonal rabbit antibody, Invitrogen) at 1:2000 dilution in 5% (w/v) non-fat milk (Carl Roth) in …

   SciScore for 10.1101/2021.12.29.21268487: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Patients are part of the COVID-19 Registry of the LMU Klinikum (CORKUM, WHO trial id DRKS00021225) and the study was approved on March 23, 2020 by the ethics committee (no. 20-245) of the Faculty of Medicine of the LMU (Ethik-Kommission
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Membranes were incubated at 7°C overnight with primary anti-spike antibody (43A11, rat IgG, Helmholtz Zentrum Mu□nchen; 1A9, mouse IgG, GeneTex; PA5-81795, polyclonal rabbit antibody, Invitrogen) at 1:2000 dilution in 5% (w/v) non-fat milk (Carl Roth) in ddH2O, washed 3 times in TBST (Tris-buffered saline with 0.1% Tween-20) and incubated at RT for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1112-035-062, goat anti-rat IgG, Jackson Immuno Research Europe; 7076S	anti-spike suggested: (Imported from the IEDB Cat# 1A9, RRID:AB_2848025)
   First, wells of a Nunc MaxiSorp plate (Thermo Fisher Scientific) were coated at RT for 5 h with 2 µg mL-1 anti-spike capture antibody (55E10, rat IgG, Helmholtz Zentrum Mu□nchen) or isotype in PBS.	anti-spike capture antibody ( 55E10 suggested: None
   Upon washing via a 100 kDa cutoff Amicon ultra filter, the samples were stained with AlexaFluor488 conjugated anti-S antibody (43A11, rat IgG, Helmholtz Zentrum Mu□nchen), diluted and afterwards analyzed in the flow cytometer.	anti-S suggested: None
   Directly coupled AlexaFluor488 labelled antibodies were generated using commercial kits (A10235, Thermo Fisher Scientific).	A10235 suggested: (ABclonal Cat# A10235, RRID:AB_2757760)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines and cell culture: HEK293T (CID3915); HEK293, S+ (CID4618); U251MG, hACE2+ (CID4663); U251MG, hACE2+, NM∼LgBiT+ (CID4697) and Vero, hACE2+ (CID4608) cells were maintained in DMEM medium (Gibco, Thermo Fisher Scientific), supplemented with 8% FBS (AC-SM-0143, Anprotec) and Pen/Strep (Gibco, Thermo Fisher Scientific).	HEK293 suggested: None U251MG suggested: None Vero suggested: None
   SARS-CoV-2 VLPs and other engineered EVs: To generate S+ VLPs containing appropriate levels of SARS-CoV-2 spike (D614G or B.1.617.2) and control-EVs, HEK293T cells were transfected using TransIT-293 (MIR2700, Mirus) according to the manufacturers protocol with carefully adjusted ratios of codon-optimized expression vectors coding for S, VSV-G, or mock together with CD63∼HiBiT or CD63∼BlaM and, optionally, M, N and E.	HEK293T suggested: None
   Antibodies REGN10987 and REGN10933 were purified from CHO cells transiently transfected with expression plasmids encoding the heavy and light chains.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   The mixture was incubated at 37°C and approximately 20,000 Vero C1008 cells (ATCC, Cat#CRL-1586) were added after 1 hour.	Vero C1008 suggested: ATCC Cat# CRL-1586, RRID:CVCL_0574)
   Virus stock preparation: CaCo-2 cells (American Type Culture Collection, ATCC, Virginia, USA) in cell culture medium (Dulbecco’s Modified Eagle’s Medium containing 2% FBS) were challenged for 2 h with a clinical isolate of SARS-CoV-2 (GISAID EPI ISL 2967222) previously obtained from a nasopharyngeal swab of a COVID-19 patient.	CaCo-2 suggested: None
   Subsequently, cell culture medium was exchanged, and three days post infection supernatants were passaged on Vero-E6 cells (ATCC, Virginia, USA).	Vero-E6 suggested: None
   Recombinant DNA
   Sentences	Resources
   SARS-CoV-2 VLPs and other engineered EVs: To generate S+ VLPs containing appropriate levels of SARS-CoV-2 spike (D614G or B.1.617.2) and control-EVs, HEK293T cells were transfected using TransIT-293 (MIR2700, Mirus) according to the manufacturers protocol with carefully adjusted ratios of codon-optimized expression vectors coding for S, VSV-G, or mock together with CD63∼HiBiT or CD63∼BlaM and, optionally, M, N and E.	VSV-G suggested: RRID:Addgene_138479)
   Software and Algorithms
   Sentences	Resources
   Data analysis was performed with GraphPad Prism 9.2.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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

   Our test overcomes the current limitations of quantitating SARS-CoV-2 NAbs as it is based on harmless, non-infectious VLPs engineered to be authentic morphological and functional mimics of SARS-CoV-2 virions. Firstly, we optimized conditions for in vitro generation of such S+ VLPs and characterized them together with a sample of heat-inactivated SARS-CoV-2 stock using standard approaches together with a novel nano flow technology. By cryo-EM, we confirmed the presence of intact, native S+ VLPs with the characteristics of coronaviruses in our preparations. Furthermore, the S+ VLPs contain SFL trimers, a molecular concentration of S very similar to authentic SARS-CoV-2 virions and particle number and ratio of S+ particles comparable to virus stocks. We calculated the number of S trimers per particle to be 27 and therefore well within the range of published figures which vary from 24 to 40 (Ke et al., 2020; Turonova et al., 2020; Yao et al., 2020). Secondly, we engineered S+ VLPs to carry a chimeric, membrane anchored and luminally oriented enzyme (BlaM) or an activator peptide (HiBiT) and generated hACE2+ Vero cells and a hACE2+ U251MG cell line, which carries hACE2+ together with an inactive, membrane-associated split reporter nLuc enzyme (LgBiT). In quantitative fusion experiments with S+ VLPs we showed that only ACE2+ cells take up S+ VLPs, while ACE2- cells were not susceptible as expected. Uptake strictly depended on spike, the viral entry factor and one of the three SARS-...

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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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   Read the original source
3. 

   Version published to 10.1101/2021.12.29.21268487v1 on medRxiv

   Dec 31, 2021