Evaluation of SARS-CoV-2 neutralization assays for antibody monitoring in natural infection and vaccine trials

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   ScreenIT

   Dec 8, 2020

   SciScore for 10.1101/2020.12.07.20245431: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Selected volunteers were invited for study participation in May 2020 and sent an electronic consent statement. IRB: This study was approved by the Fred Hutch Institutional Review Board and all study materials were provided in both English and Spanish
   Randomization	In the first phase, study volunteers were randomly selected for participation after stratification by ZIP code, and within ZIP code, age, gender and race/ethnicity.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The assay is a chemiluminescent microparticle immunoassay …

   SciScore for 10.1101/2020.12.07.20245431: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: Selected volunteers were invited for study participation in May 2020 and sent an electronic consent statement. IRB: This study was approved by the Fred Hutch Institutional Review Board and all study materials were provided in both English and Spanish
   Randomization	In the first phase, study volunteers were randomly selected for participation after stratification by ZIP code, and within ZIP code, age, gender and race/ethnicity.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The assay is a chemiluminescent microparticle immunoassay that measures IgG antibodies to the SARS-CoV-2 nucleocapsid protein.	SARS-CoV-2 nucleocapsid protein . suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Protein antigens: A recombinant form of a synthetic construct (SARS_CoV_2_ectoCSPP (26); GenBank: QJE37812.1) of the spike (S) glycoprotein from SARS-CoV-2 Wuhan-Hu-1 was produced in human HEK293 cells (FreeStyle™ 293-F Cells, ThermoFisher, Waltham, MA) using a lentivirus expression system (27) and purified by nickel affinity and size-exclusion chromatography.	HEK293 suggested: RRID:CVCL_6642)
   Twenty-four hours prior infection with VSV(G*ΔG-luciferase), 293T cells were transfected with pcDNA-WuhanCoV-S-D19.	293T suggested: None
   Pseudovirions were produced in HEK 293T/17 cells (catalog number CRL-11268; ATCC) by transfection using Fugene 6 (catalog number E2692; Promega).	HEK 293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   Vero E6 cells were seeded at 2×104 cells/well in a 96-well plate 24 h before the assay.	Vero E6 suggested: None
   After incubation, plasma-virus mixture was transferred onto the Vero cells.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   s neutralization assays: Neutralization of SARS-CoV-2 Spike-pseudotyped virus was performed by using lentiviral vectors and infection in either HEK 293T cells expressing human ACE2 (293T/ACE2.MF) or TZM-bl cells expressing both ACE2 and TMPRSS2 (TZM-bl/ACE2/TMPRSS2 cells).	HEK 293T suggested: None TZM-bl/ACE2/TMPRSS2 suggested: None
   293T/ACE2 cells pseudovirus assay: For the 293T/ACE2 assay, a pre-titrated dose of virus was incubated with serial 3-fold dilutions of test sample in duplicate in a total volume of 150 ul for 1 hr at 37°C in 96-well flat-bottom black/white culture plates.	293T/ACE2 suggested: RRID:CVCL_YZ65)
   ACE2/TMPRSS2 TZM-bl cells pseudovirus assay: For the TZM-bl/ACE2/TMPRSS2 assay, a pre-titrated dose of virus was incubated with serial 3-fold dilutions of test sample in duplicate in a total volume of 150 ul for 1 hr at 37°C in 96-well flat-bottom culture plates.	TZM-bl suggested: None
   Software and Algorithms
   Sentences	Resources
   One aliquot was submitted for Architect SARS-CoV-2 IgG assay (Abbott).	Abbott suggested: (Abbott, RRID:SCR_010477)
   Patient demographic information (sex and age) was extracted from a RedCap survey database.	RedCap suggested: (REDCap, RRID:SCR_003445)

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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: We detected the following sentences addressing limitations in the study:

   ELISA-based assays have two major limitations: i) inability to account for synergistic action of antibodies targeting different epitopes; and ii) detection only of antibodies that block interaction between RBD and ACE2, thus omitting antibodies that neutralize virus via non-RBD sites on the virus glycoprotein (24, 40). For example, synergistic action of antibodies against RBD and the S2 domain has been reported (41). There are two ways of performing such a surrogate assay: soluble biotinylated ACE2 competing with serum antibodies for binding to immobilized RBD or spike, or an opposite version with soluble RBD and immobilized ACE2 (21). Abe et al. found that an assay with soluble ACE2 and immobilized RBD was more sensitive and yielded ND50 values that correlated with ND50 titers obtained in the classical cell-based PRNT with a coefficient of determination of 0.6. The GenScript assay that we have tested in our study utilizes immobilized ACE2, which likely explains why we were not able to measure ND50 titers for the majority of samples. Of note, samples used by Abe et al. were also collected from mild-to-moderate COVID-19 patients. In conclusion, a surrogate assay can be used cautiously as an alternative to cell-based assays to obtain preliminary qualitative results, to rapidly distinguish between samples with high and low neutralizing potency, and when a cell-based assay is not available or reasonably feasible.

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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 10.1101/2020.12.07.20245431v1 on medRxiv

   Dec 8, 2020