Epigenome-wide DNA methylation profiling of healthy COVID-19 recoverees reveals a unique signature in circulating immune cells

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1. 

   Version published to 10.1101/2021.07.05.21260014v2 on medRxiv

   Nov 8, 2021
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   ScreenIT

   Jul 8, 2021

   SciScore for 10.1101/2021.07.05.21260014: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: For samples from the natural exposure cohort, all participants provided written informed consent, and the present study was approved by the Regional Ethics Committee for Human Research in Linköping (Dnr. 2019-0618). IRB: For samples from the natural exposure cohort, all participants provided written informed consent, and the present study was approved by the Regional Ethics Committee for Human Research in Linköping (Dnr. 2019-0618).
   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
   Cons were defined as neither having any positive …

   SciScore for 10.1101/2021.07.05.21260014: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: For samples from the natural exposure cohort, all participants provided written informed consent, and the present study was approved by the Regional Ethics Committee for Human Research in Linköping (Dnr. 2019-0618). IRB: For samples from the natural exposure cohort, all participants provided written informed consent, and the present study was approved by the Regional Ethics Committee for Human Research in Linköping (Dnr. 2019-0618).
   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
   Cons were defined as neither having any positive circulating IgG-antibody or T cell responses to SARS-CoV-2, while CC19s were defined by the presence of SARS-CoV-2-specific IgG antibodies in plasma using suspension multiplex immunoassay (SMIA), some of which were positive for IgG in saliva, rapid test and in T cell responses as well.	SARS-CoV-2-specific IgG suggested: None
   Stimulation with anti-CD3 antibody at a concentration of 1 µg/ml was used as a positive control for each subject.	anti-CD3 suggested: None
   Anti-CD28 antibody (3608-1-50, Mabtech, Sweden) was included at a final concentration of 0.1 μg/ml as a co-stimulator.	Anti-CD28 suggested: None
   The beads were resuspended in 100 µl of 1 µg/ml goat anti-human IgG-PE labelled antibody (Southern BioTech,, Birmingham, AL, USA. Cat. #2040-09) in PBS-T + 1% BSA and incubated for 30 min at RT in the dark with rotation at 600 rpm.	anti-human IgG-PE suggested: (SouthernBiotech Cat# 2040-09, RRID:AB_2795648)
   Next, the cells were incubated with mouse-anti-dsRNA antibody (Scions, Code: J2 at 1:100 dilution) for 1.5 h followed by detection using horseradish peroxidase–conjugated goat anti-mouse IgG (heavy plus light chain) (Catalog: 1706516, Bio-Rad Laboratories, Hercules, CA, USA) (1:1000) for 1 h.	mouse-anti-dsRNA suggested: None anti-mouse IgG suggested: (Bio-Rad Cat# 170-6516, RRID:AB_11125547)
   Experimental Models: Cell Lines
   Sentences	Resources
   SARS-CoV-2 or mock Vero cell supernatant was added to the PBMC cultures corresponding to a multiplicity of infection of 0.01.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Finally, the SARS CoV-2 infected Vero E6 cells were identified using 3-aminoethylcarbazole (AEC) substrate.	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   Next, the cells were incubated with mouse-anti-dsRNA antibody (Scions, Code: J2 at 1:100 dilution) for 1.5 h followed by detection using horseradish peroxidase–conjugated goat anti-mouse IgG (heavy plus light chain) (Catalog: 1706516, Bio-Rad Laboratories, Hercules, CA, USA) (1:1000) for 1 h.	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Concentrations of extracted DNA were measured using the Qubit® 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, U.S), using dsDNA High Sensitivity (HS) Assay Kit and RNA HS Assay Kit.	Qubit® suggested: None
   Thereafter, we performed singular value decomposition (SVD) analyses using the ChAMP package (45	ChAMP suggested: (ChAMP, RRID:SCR_012891)
   Differential DNA methylation analysis in vivo: As we were interested in studying CpGs that were differentially methylated between CC19s and non-infected controls from both before and after the start of the COVID-19 pandemic, we performed differential DNA methylation analyses, using the limma package (version 3.46.0).	limma suggested: (LIMMA, RRID:SCR_010943)
   Pathway over-representation analyses: To make biological sense of the putatively SARS-CoV-2-induced DNA methylation differences, we performed PANTHER pathway over-representation test analyses using the PANTHER database (version 16.0).	PANTHER suggested: (PANTHER, RRID:SCR_004869)
   The significantly enriched pathways were displayed in dot plots generated in R using ggplot2 package (version 3.3.3).	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
   The graph clustering algorithm MCODE (48) was used to identify molecular complexes and create a large disease module, which was then fitted to a protein-protein interaction network, and both were analysed and rendered in Cytoscape (version 3.8.0).	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)
   In addition, the inference of modules was performed with two other methods from the MODifieR package (DIAMOnD and WGCNA)(50) to study whether it was possible to condense the module genes to fewer genes of particular interest within the network, for both the in vivo and the in vitro setting.	DIAMOnD suggested: (DIAMOND, RRID:SCR_009457)
   Overlap to SARS-CoV-2 interactome: A publicly available protein-protein interaction (PPI) network of SARS-COV-2 and human genes curated by BioGRID (version 4.4.197) was downloaded from the Network Data Exchange in Cytoscape (version 3.8.0).	BioGRID suggested: (BioGrid Australia, RRID:SCR_006334)

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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:

   Although an obvious limitation of the study is the lack of validation of the DNAm findings on a transcriptional level, it serves as a pilot study that generates hypotheses for further studies within the field. Hence, whether the observed DNAm patterns are indeed associated or even causally linked to host protective or host detrimental immune responses still needs to be addressed in future studies. With more well-designed, larger, consecutive sample materials, possibly also in closer proximity to the time of infection with SARS-CoV-2, it will be possible to study the role of DNAm alterations in anti-viral defence and in viral manipulation of the same. An advantage of the investigation of epigenetic modifications in in mild to moderately ill patients, is that we may be able to discern DNAm differences that otherwise would have been masked due to an overriding inflammatory response. These subtle changes may not only be relevant to how a less severe immune response is mounted towards SARS-CoV-2, but also in the case of long-COVID-19. The presentation of longstanding symptoms could be caused by detrimentally changed DNAm patterns, originally triggered as a short-term anti-viral response. This should be explored in detail in further studies since the risk is that these short-term responses may permanently alter and erroneously manifest in the DNA methylome.

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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.
   • Thank you for including a protocol registration statement.

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

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3. 

   Version published to 10.1101/2021.07.05.21260014v1 on medRxiv

   Jul 6, 2021