Immune defects associated with lower SARS-CoV-2 BNT162b2 mRNA vaccine response in aged people

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1. Version published to 10.1172/jci.insight.161045

   Sep 8, 2022
2. 

   ScreenIT

   Jan 11, 2022

   SciScore for 10.1101/2022.01.07.22268806: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: ETHICS STATEMENT: Participants gave written consent and the study was approved by the Ethics Committee of the Virgen del Rocio University Hospital (protocol code “COVIMARATON_2020”; internal code 0156-N-21). IRB: ETHICS STATEMENT: Participants gave written consent and the study was approved by the Ethics Committee of the Virgen del Rocio University Hospital (protocol code “COVIMARATON_2020”; internal code 0156-N-21).
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Three young subjects were excluded from the study due to a positive result for SARS-CoV-2 PCR or SARS-CoV-2 …

   SciScore for 10.1101/2022.01.07.22268806: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: ETHICS STATEMENT: Participants gave written consent and the study was approved by the Ethics Committee of the Virgen del Rocio University Hospital (protocol code “COVIMARATON_2020”; internal code 0156-N-21). IRB: ETHICS STATEMENT: Participants gave written consent and the study was approved by the Ethics Committee of the Virgen del Rocio University Hospital (protocol code “COVIMARATON_2020”; internal code 0156-N-21).
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Three young subjects were excluded from the study due to a positive result for SARS-CoV-2 PCR or SARS-CoV-2 RBD-specific antibodies prior to vaccination.	SARS-CoV-2 RBD-specific suggested: None
   The stimulation was performed in the presence of 10 µg/mL of brefeldin A (Sigma Chemical Co) and 0.7 µg/mL of monensin (Golgi Stop, BD Biosciences) protein transport inhibitors, anti-CD107a-BV650 (clone H4A3; BD Biosciences) monoclonal antibody and purified CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences) as previously described (Ferrando-Martinez et al., 2012; Pérez-Gómez et al., 2021).	anti-CD107a-BV650 suggested: None CD28 suggested: None CD49d suggested: (Abcam Cat# ab115904, RRID:AB_10903696)
   For ex vivo DC phenotyping, PBMCs were extracellularly stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), anti-CD11c-BV650 (clone B-ly6), anti-HLA-DR-BV711 (clone G46-6), anti-Lin-2-FITC, anti-CD16-BV605 (clone 3G8), anti-CCR7-BV786 (CD197) (clone 3D12), anti-CD86-BV421 (clone 2331 (FUN-1)), anti-PD-L1 PeCF594 (CD274) (clone MIH1), anti-Integrin-β7-APC (clone FIB504) (BD Biosciences), anti-CD4-PerCPCy5,5 (clone OKT4), anti-CD1c-APCCy7 (clone L161), anti-CD141-PeCy7 (clone M80) (BioLegend) and anti-CD123-AF700 (clone 32703) (R&D Systems) antibodies.	anti-CD11c-BV650 suggested: None anti-HLA-DR-BV711 suggested: None anti-Lin-2-FITC suggested: None anti-CD16-BV605 suggested: None anti-CCR7-BV786 suggested: None CD197 suggested: None anti-CD86-BV421 suggested: None anti-PD-L1 suggested: None CD274 suggested: None anti-Integrin-β7-APC suggested: None anti-CD4-PerCPCy5,5 suggested: None anti-CD1c-APCCy7 suggested: None anti-CD141-PeCy7 suggested: None anti-CD123-AF700 suggested: None
   Human serum standard reference material of anti-SARS-CoV-2 immunoglobulin (first WHO International Standard and International Reference Panel for anti-SARS-CoV-2 immunoglobulin from NIBSC, UK, NIBSC code: 20/150) was used as standard curve to titer anti-SARS-CoV-2 IgG antibody in plasma samples.	anti-SARS-CoV-2 immunoglobulin suggested: None
   Secondary antibodies, streptavidin-horseradish peroxidase-conjugated mouse anti-human IgG (Hybridoma Reagent Laboratory, Baltimore, MD,) was used at a 1:5,000 dilution in 1% milk containing 0.05% Tween-20 in PBS.	anti-human IgG suggested: (LSBio (LifeSpan Cat# LS-C6452-25, RRID:AB_834632)
   Anti-SARS-CoV-2 IgG antibody titers for each donor were calculated as Binding Antibody Units (BAU)/ml according to the manufacturers’ information regarding the WHO Standard and was determined based on sigmoidal dose-response nonlinear regression, 4PL, using GraphPad Prism, version 8.0 (GraphPad Software, Inc.).	Anti-SARS-CoV-2 IgG suggested: None
   Software and Algorithms
   Sentences	Resources
   All these immune parameters were correlated with SARS-CoV-2 specific humoral and T cell responses after the vaccination.	SARS-CoV-2 suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
   For T cell phenotyping, cells were extracellularly stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), anti CD8-PerCP-Cy5.5 (clone SK1), anti-CD45RA-PeCy7 (clone L48), anti-CD3-BV711 (clone SP34-2) (BD Bioscience), anti-HLA-DR-BV570 (clone L243), anti CD161-BV421 (clone HP-3G10) (BioLegend); permeabilized and fixed with Fixation/Permeabilization buffer (eBioscience™); and intracellularly stained with: anti-Ki67 FITC (clone 11F6) (BioLegend).	BioLegend) suggested: (BioLegend, RRID:SCR_001134)
   To assay monocytes functionality, PBMCs were extracellularly stained with LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Life Technologies), anti-DUMP-channel-V450 (CD3 clone SP34-2, CD19 clone HIB19, CD20 clone L27, CD56 clone B159), anti-CD14-BV650 (clone M5E2), anti-CD16-PeCF594 (clone 3G8), anti-HLA-DR-BV570 (clone L243), (BioLegend); permeabilized and fixed with BD Cytofix/CytoPerm (BD Biosciences); and intracellularly stained with anti-IL-6-Pe (clone MQ2-6A3), anti-IL-1α-FITC (clone AS5), anti-TNF-α-AF700 (clone MAb11) (BD Biosciences).	BD Biosciences) suggested: None
   Multiparametric flow cytometry were performed on an LRS Fortessa flow cytometer using FACS Diva software (BD Biosciences), acquiring 0.5-1 × 106 events.	BD Biosciences suggested: (BD Biosciences, RRID:SCR_013311)
   Data were analyzed using the FlowJo 10.7.1 software (Treestar, Ashland, OR).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Anti-SARS-CoV-2 IgG antibody titers for each donor were calculated as Binding Antibody Units (BAU)/ml according to the manufacturers’ information regarding the WHO Standard and was determined based on sigmoidal dose-response nonlinear regression, 4PL, using GraphPad Prism, version 8.0 (GraphPad Software, Inc.).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   The program used for determining the sj/β TREC ratio was Bio-Rad QuantaSoft software v.1.7.4.	QuantaSoft suggested: None

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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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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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 pages 34 and 38. 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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.01.07.22268806v1 on medRxiv

   Jan 8, 2022