High Incidence of SARS-CoV-2 Variant of Concern Breakthrough Infections Despite Residual Humoral and Cellular Immunity Induced by BNT162b2 Vaccination in Healthcare Workers: A Long-Term Follow-Up Study in Belgium

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1. Version published to 10.3390/v14061257

   Jun 9, 2022
2. 

   ScreenIT

   Jan 20, 2022

   SciScore for 10.1101/2022.01.17.22269081: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Healthcare workers were included after signing an informed consent form. IRB: This trial was performed according to the declaration of Helsinki and was approved by both the local Ethical Committee of the AZ Groeninge hospital (B3962021000022) and the Belgium Federal Agency of Drugs and Health Products (FAGG; protocol no. AZGS2021005).
   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
   Serum anti-S IgA antibodies were measured with the Anti-SARS-CoV-2 IgA enzyme immunoassay from EUROIMMUN (Lübeck, Germany) on an ETI-Max 3000 instrument …

   SciScore for 10.1101/2022.01.17.22269081: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: Healthcare workers were included after signing an informed consent form. IRB: This trial was performed according to the declaration of Helsinki and was approved by both the local Ethical Committee of the AZ Groeninge hospital (B3962021000022) and the Belgium Federal Agency of Drugs and Health Products (FAGG; protocol no. AZGS2021005).
   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
   Serum anti-S IgA antibodies were measured with the Anti-SARS-CoV-2 IgA enzyme immunoassay from EUROIMMUN (Lübeck, Germany) on an ETI-Max 3000 instrument from DiaSorin (Saluggia, Italy).	anti-S IgA suggested: None Anti-SARS-CoV-2 IgA suggested: None
   Sample concentrations were determined using the anti-RBD calibrator antibody and WHO International Standard Serum	anti-RBD suggested: None
   2.2.3 Anti-N IgG assay: The presence of serum anti-N IgG antibodies was determined via the Anti-SARS-CoV-2-NCP (IgG) enzyme immunoassay from EUROIMMUN on an ETI-Max 3000 instrument from DiaSorin.	anti-N IgG suggested: None Anti-SARS-CoV-2-NCP ( IgG suggested: None
   Two hours later, the medium was replaced by medium containing anti-VSV-G antibody (I1-hybridoma, ATCC CRL-2700) to neutralize residual VSV-G input.	anti-VSV-G suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, HEK-293T cells (SARS-CoV-2) were transfected with the respective S protein expression plasmids, and one day later infected (MOI = 2) with green fluorescent protein (GFP)-encoding VSVΔG backbone virus (purchased from Kerafast).	HEK-293T suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
   To quantify neutralizing antibodies (nAbs), serial dilutions of serum samples were incubated for 1 h at 37 °C with an equal volume of S pseudotyped VSV particles and inoculated on Vero E6 cells (SARS-CoV-2) for 18 h.	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Genome assembly was performed using the ARTIC bioinformatics pipeline v1.1.3, which entails adapter trimming, mapping to the reference strain Wuhan-Hu-1 (MN908947), as previously described (36).	Wuhan-Hu-1 suggested: None
   Software and Algorithms
   Sentences	Resources
   This figure was created using Biorender (www.biorender.com). 2.1.3 Serum and peripheral blood mononuclear cell (PBMC) isolation: Venous blood from three K2-EDTA tubes was used to isolate PBMCs via density gradient isolation with Ficoll (lymphoprep™, STEMCELL technologies, Norway) using SepMate tubes (STEMCELL technologies, Norway).	Biorender suggested: (Biorender, RRID:SCR_018361)
   Neutralization IC50 values were determined by normalizing the serum neutralization dilution curve to a virus (100%) and cell control (0%) and fitting in GraphPad Prism (inhibitor vs. response, variable slope, four parameters model with top and bottom constraints of 100 % and 0 % respectively). 2.4 Stimulation of SARS-CoV-2 specific T cells: Heparinized whole blood was used for the EUROIMMUN SARS-CoV-2 interferon gamma release assay (IGRA) kit that was executed according to the manufacturer’s instructions adapted as described below (Supplementary Figure 1).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The libraries were sequenced on a MinION using R9.4.1 flow-cells (Oxford Nanopore Technologies, Oxford, UK) and MinKnow software v21.02.1.	MinION suggested: (MinION, RRID:SCR_017985) MinKnow suggested: None
   2.7 Statistical analyses: Statistical analysis was performed using both Microsoft Excel (version 365 for Windows, Microsoft Corporation, USA) and GraphPad Prism (version 9.0.0 for Windows, GraphPad Software, USA)	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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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:

   At last, it must be noted that this trial has several limitations. Firstly, the number of subjects is rather limited and was based on a power analysis for the serological and IFN-γ release read-outs. Hence, this design is less suited to pick up rather unexpected events such as BTI. Larger studies that look at both arms of the adaptive immune response months after vaccination are needed to further finetune our findings and to characterize BTI more reliable. Secondly, only SARS-CoV-2 naive individuals were included in this study. The pronounced recall response due to vaccination in convalescent individuals has been described and even led to the discussion whether these individuals should receive one rather than two vaccine doses. Addressing this was beyond our scope as we specifically aimed to study the BNT162b2 vaccine-induced de novo immune response in a real-world setting. Thirdly, the emergence of new and potentially more dangerous SARS-CoV-2 variants leads the binding and neutralization assays available at any given time. In this perspective, running neutralization assays with VoC RBD could give deeper insights in the breadth of the vaccine-induced functional immunity. Finally, long-term sustainability of the vaccine-induced immune response can only be truly considered when looking even beyond the three month timepoint. This will be particularly relevant as we are slowly progressing from a global pandemic state to a genuine endemic circulation of the SARS-CoV-2 virus.

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

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3. 

   Version published to 10.1101/2022.01.17.22269081v1 on medRxiv

   Jan 17, 2022