Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy.
Article activity feed
-
SciScore for 10.1101/2020.08.13.20157222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Sera acquisition: All patient sera were acquired after approval by the respective institutional review boards and/or equivalent oversight bodies (Bioethics Committee, Independent Ethics Committee) are indicated: (1) Mount Sinai Hospital Institutional Review Board (New York, USA), (2) Louisiana State University Health Sciences Center – Shreveport (LSUHS, Louisiana, USA), and (3) Fundacion Instituto Leloir-CONICET, Universidad Nacional de San Martin, Laboratorio Lemos SRL, Universidad de Buenos Aires (COVIDAR Argentina Consortium, Buenos Aires, Argentina).
Consent: All necessary patient/participant consent has been obtained and the appropriate institutional …SciScore for 10.1101/2020.08.13.20157222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Sera acquisition: All patient sera were acquired after approval by the respective institutional review boards and/or equivalent oversight bodies (Bioethics Committee, Independent Ethics Committee) are indicated: (1) Mount Sinai Hospital Institutional Review Board (New York, USA), (2) Louisiana State University Health Sciences Center – Shreveport (LSUHS, Louisiana, USA), and (3) Fundacion Instituto Leloir-CONICET, Universidad Nacional de San Martin, Laboratorio Lemos SRL, Universidad de Buenos Aires (COVIDAR Argentina Consortium, Buenos Aires, Argentina).
Consent: All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.Randomization not detected. 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 Antibodies against SARS-CoV2 (2B3E5 from Dr. Thomas Moran and GTX632604 from GeneTex), ACE2 (66699-1-Ig from Proteintech and Rb ab108252 from abcam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used. SARS-CoV2suggested: NoneGTX632604suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)ACE2suggested: (Abcam Cat# ab108252, RRID:AB_10864415)VSV-Gsuggested: Noneanti-HAsuggested: (Novus Cat# NB600-363, RRID:AB_10001504)For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. anti-mousesuggested: Noneanti-rabbitsuggested: NoneIgG and IgM antibodies were detected with secondary antibodies conjugated to HRP (Millipore AP101P for anti-Human IgG and Invitrogen A18841 for anti-Human IgM). IgMsuggested: (Millipore Cat# AP101P, RRID:AB_92409)anti-Human IgGsuggested: (Thermo Fisher Scientific Cat# A18841, RRID:AB_2535618)anti-Human IgMsuggested: NoneForty-eight hours post infection, cells were fixed in 10% PFA and stained with mouse anti-SARS-CoV nucleoprotein antibody. anti-SARS-CoV nucleoproteinsuggested: NoneExperimental Models: Cell Lines Sentences Resources 293T and Vero-CCL81 cells were selected with 2 or 10μg/mL of puromycin, respectively. 293Tsuggested: NoneTo generate ACE2 and TMPRSS2 expressing 293T cells, 293T-ACE2 cells were transduced with the VSV-G pseudotyped lentivirus packaging TMPRSS2. 293T-ACE2suggested: RRID:CVCL_YZ65)Briefly, ~600 50% tissue culture infectious doses (TCID50) of virus was incubated with a serial dilution of patient sera for 1hr at 37°C prior to infection of Vero-E6 cells. Vero-E6suggested: NoneFor the VNAs performed in our lab (ISMMS-1), a pre-titrated amount of pseudotyped particles (diluted to give approximately 105 RLU) was incubated with a 4-fold serial dilution of patient sera for 30 minutes at room temperature prior to infection of Vero-CCL81 cells seeded the previous day. Vero-CCL81suggested: NoneSoftware and Algorithms Sentences Resources VSV-G pseudotyped lentiviruses packaging ACE2 or TMPRSS2 expression constructs were generated by using Bio-T (Bioland; B01-01) to transfect 293T cells with the second-generation lentiviral packaging plasmid (Addgene; 12259), pCAGG-VSV-G, and the desired expression construct (i.e. ACE2 or TMPRSS2). Addgenesuggested: (Addgene, RRID:SCR_002037)Background was subtracted from the OD values, samples were determined to be positive if ≥ 3 fold over the negative control and AUC was calculated in PRISM. PRISMsuggested: (PRISM, RRID:SCR_005375)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).
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.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.
- Thank you for including a protocol registration statement.
-
-
-
SciScore for 10.1101/2020.08.13.20157222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Sera acquisition All patient sera were acquired after approval by the respective institutional review boards and/or equivalent oversight bodies (Bioethics Committee, Independent Ethics Committee) are indicated: (1) Mount Sinai Hospital Institutional Review Board (New York, USA), (2) Louisiana State University Health Sciences Center – Shreveport (LSUHS, Louisiana, USA), and (3) Fundacion Instituto Leloir-CONICET, Universidad Nacional de San Martin, Laboratorio Lemos SRL, Universidad de Buenos Aires (COVIDAR Argentina Consortium, Buenos Aires, Argentina). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not … SciScore for 10.1101/2020.08.13.20157222: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Sera acquisition All patient sera were acquired after approval by the respective institutional review boards and/or equivalent oversight bodies (Bioethics Committee, Independent Ethics Committee) are indicated: (1) Mount Sinai Hospital Institutional Review Board (New York, USA), (2) Louisiana State University Health Sciences Center – Shreveport (LSUHS, Louisiana, USA), and (3) Fundacion Instituto Leloir-CONICET, Universidad Nacional de San Martin, Laboratorio Lemos SRL, Universidad de Buenos Aires (COVIDAR Argentina Consortium, Buenos Aires, Argentina). Randomization not detected. 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 Antibodies against SARS-CoV2 (2B3E5 from Dr. Thomas Moran and GTX632604 from GeneTex), ACE2 (66699-1-Ig from Proteintech and Rb ab108252 from abcam), VSV-G (A00199 from Genescript), VSV-M (EB0011 from Kerafast), anti-HA (NB600-363 from Novus), and CoX IV (926-42214 from LI-COR) were used. SARS-CoV2suggested: (EnCor Biotechnology Cat# MCA-2G1, RRID:AB_2861173)GTX632604suggested: NoneACE2suggested: (Abcam Cat# ab108252, RRID:AB_10864415)VSV-Gsuggested: Noneanti-HAsuggested: (Novus Cat# NB600-363, RRID:AB_10001504)For secondary staining, membranes were washed and incubated with the appropriate Alexa Fluor 647-conjugated anti-mouse antibody or Alexa Fluor 647-conjugated anti-rabbit antibody. anti-mousesuggested: Noneanti-rabbitsuggested: NoneIgG and IgM antibodies were detected with secondary antibodies conjugated to HRP (Millipore AP101P for anti-Human IgG and Invitrogen A18841 for anti-Human IgM). IgMsuggested: (Millipore Cat# AP101P, RRID:AB_92409)anti-Human IgGsuggested: (Thermo Fisher Scientific Cat# A18841, RRID:AB_2535618)anti-Human IgMsuggested: NoneForty-eight hours post infection, cells were fixed in 10% PFA and stained with mouse anti-SARS-CoV nucleoprotein antibody. anti-SARS-CoV nucleoproteinsuggested: NoneExperimental Models: Cell Lines Sentences Resources To generate ACE2 and TMPRSS2 expressing 293T cells, 293T-ACE2 cells were transduced with the VSV-G pseudotyped lentivirus packaging TMPRSS2. 293T-ACE2suggested: NoneBriefly, ~600 50% tissue culture infectious doses (TCID50) of virus was incubated with a of patient sera for 1hr at 37ºC prior to infection of Vero-E6 cells. Vero-E6suggested: NoneData is presented as mean +/- SEM from two independent experiments done in technical triplicates. *, CoV2pp were used to infect Vero- CCL81 cells in the presence of a 4-fold of patient sera as described in the Methods. Vero- CCL81suggested: NoneFor statistics, ns = not significant, ** is a p-value <0.01, and **** is a p-value <0.0001. ( B) Western blot of ACE2 expression in 293T cell lines. 293Tsuggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)Software and Algorithms Sentences Resources VSV-G pseudotyped lentiviruses packaging ACE2 or TMPRSS2 expression constructs were generated by using Bio-T (Bioland; B01-01) to transfect 293T cells with the second-generation lentiviral packaging plasmid (Addgene; 12259), pCAGG-VSV-G, and the desired expression construct (i.e. ACE2 or TMPRSS2). Addgenesuggested: (Addgene, RRID:SCR_002037)Background was subtracted from the OD values, samples were determined to be positive if ≥3 fold over the negative control and AUC was calculated in PRISM. PRISMsuggested: (PRISM, RRID:SCR_005375)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).
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.
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: We did not find any issues relating to colormaps.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
-