SARS–CoV–2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro
Jiang H, Mei YF.
- DOI
- 10.3390/v13102056
- Record issued
- 2026-08-05
- Engine
- 7.15.0
- Exported
- 2026-09-20
Prepared by Alpha1. This document is confidential: it is intended for the recipient it was shared with and must not be redistributed. The live record at alpha1science.com/verify/0e079a22-b4c4-4fea-a809-111f5ffe60d8 is authoritative.
How this rating was calculated
- IntegrityIntegrity concern ×2−1★
- ClaimsOverstated claim−0.5★
- ReportingStudy design not met−0.5★
- ReportingKey resources not met−0.5★
- ReportingData & code availability not met−0.5★
- ReportingStatistical analysis partially met−0.25★
- Statistics were not checked: no recomputable values were found in this text — no test statistic reported with its degrees of freedom, no effect estimate printed with both a 95% CI and a p-value, and no percentage printed with both its count and its denominator.
- 01Study design lacks key rigor safeguards
The study lacks adequate reporting of randomization, blinding (except for scoring), power analysis, inclusion/exclusion criteria, outlier handling, and independent replication, while only reporting replicate distinction and controls adequately.
“All scoring was performed under blinded conditions.”
Methods - 02Key resources not identified
Key resources are inadequately identified: antibodies are listed with catalog numbers but without RRIDs or validation data, cell lines are not authenticated, mycoplasma testing is not reported, and software versions are incomplete.
“Data were analyzed by Student’s t test or ANOVA followed by Tukey’s multiple-comparison tests using GraphPad 8.”
Methods - 03Data and code not shared
The data availability statement is vague (data available in the main text and supplementary), and no raw data are deposited in a public repository. Custom code is not shared.
“The data presented in this study are available in the main text and .”
Data availability - 04Conclusion reaches beyond the evidence
Full-length spike-based vaccines may have side effects by inhibiting adaptive immunity.
“our findings also imply a potential side effect of the full–length spike–based vaccine”
Discussion
This Kaimen Rigor review uses Kaimen Rigor reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.
The paper has a well-articulated premise, detailed methods, appropriate controls and independent biological replicates, and transparent funding/COI disclosure — the strengths of this in vitro study. Its main weaknesses are a truncated data-availability statement with no repository, under-reported study-design elements (power analysis, blinding outside IF scoring, outlier handling, independent replication), missing cell-line authentication/mycoplasma statements, threshold-only p-values with no verifiable recomputation, and one overstated vaccine-related implication.
Synthesis of three independent reviewer runs of the same model (sampling stability, not corroboration by different systems), plus a copyedit pass and verification components: 29 references checked (0 retracted, 0 not found in registry), 0 statistics recomputed (all p-values threshold-only), 1 reproducibility link live, no preregistration. Reviewers diverged on study design (fail vs warn), biological variables (pass vs N/A), key resources (pass/warn/fail), and statistics (warn vs fail); each divergence was resolved at the sub-criterion level and both positions are preserved in the dimension details.
Numerical inconsistencies
1 finding · worst lowValues that contradict each other or are impossible for the stated sample: recomputed p-values and test statistics, GRIM/GRIMMER checks on summary numbers, percentages against their own counts, totals against their parts, and estimates against their own confidence intervals.
- Internal contradictions in the reported numbersAssessed
- lowinternal contradictionThe comet assay description states '20 fields with approximately 100 cells in each sample' implying 2000 cells, but the quantification says 'n > 200 comets'. This is inconsistent.
Twenty fields with approximately 100 cells in each sample were evaluated and quantified using the Fiji software to determine the tail length (tail moment). ... Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of three independent experiments.
Figure 2reviewer’s wording - lowinternal contradictionThe significance notation in Figure 2 legend is contradictory: 'NS (Not Significant): * p > 0.05' uses an asterisk for a non-significant result, which is non-standard.
“NS (Not Significant): * p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.”
Figure 2
Overstated conclusions
2 findings · worst mediumConclusions that reach past what the paper's own results support — including a significance claim that no longer holds when the statistic is recomputed, and efficacy resting on an unvalidated surrogate endpoint.
- Conclusions overstated beyond the evidenceAssessed
- Conclusions only partially backed by the presented evidenceAssessed
11 major claims checked against the paper's own evidence: 1 not fully backed by the presented evidence (unsupported or overstated).
- overstatedReviewer 2Full-length spike-based vaccines may have side effects by inhibiting adaptive immunity.The in vitro findings are suggestive but do not directly demonstrate in vivo vaccine effects; the claim is extrapolated beyond the data.Evidence: The paper only shows in vitro cell line data; no vaccine or in vivo data are presented.
“our findings also imply a potential side effect of the full–length spike–based vaccine”
Discussion - partialReviewer 1Full-length spike-based vaccines may have potential side effects due to inhibition of V(D)J recombination.The paper demonstrates in vitro effects on V(D)J recombination but does not test in vivo vaccine effects. The conclusion is an extrapolation, acknowledged as a 'potential' side effect.Evidence: Figure 4, Discussion.
“underscore the potential side effects of full-length spike-based vaccines”
Discussion - partialReviewer 3Full-length spike-based vaccines may inhibit V(D)J recombination in B cells.The in vitro evidence suggests a potential mechanism, but the claim is extrapolated to in vivo vaccine effects without direct evidence. The paper acknowledges this is speculative.Evidence: Discussion mentions that the finding is consistent with a study showing lower antibody titers with full-length spike vaccine.
Our data provide valuable details on the involvement of spike protein subunits in DNA damage repair, indicating that full-length spike-based vaccines may inhibit the recombination of V(D)J in B cells, which is also consistent with a recent study that a full-length spike-based vaccine induced lower antibody titers compared to the RBD-based vaccine [].
Discussionreviewer’s wording - supportedReviewers 1, 3SARS-CoV-2 spike protein significantly inhibits DNA damage repair.The paper provides direct evidence from HR and NHEJ reporter assays and comet assays, showing reduced repair in the presence of spike protein.Evidence: Figure 1B-E, Figure 2B-G, and associated statistical analysis.
“SARS–CoV–2 spike protein significantly inhibits DNA damage repair”
Abstract - supportedReviewers 1, 3Spike protein localizes in the nucleus.Immunofluorescence and subcellular fractionation data show spike protein in the nuclear and chromatin-bound fractions.Evidence: Figure 1A, Figure 3A.
“the spike protein localizes in the nucleus”
Abstract - supportedReviewer 1Spike protein inhibits DNA damage repair by impeding BRCA1 and 53BP1 recruitment.Foci formation assays show reduced BRCA1 and 53BP1 foci in spike-expressing cells after DNA damage.Evidence: Figure 3D-G.
“inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site”
Abstract - supportedReviewer 1Spike protein inhibits V(D)J recombination in vitro.The V(D)J reporter assay shows reduced GFP-positive cells when spike is expressed, indicating impaired recombination.Evidence: Figure 4A-C.
“Spike Protein Impairs V(D)J Recombination In vitro”
Abstract - supportedReviewer 2SARS-CoV-2 spike protein inhibits DNA damage repair in vitro.The claim is supported by head-to-head comparisons of NHEJ and HR reporter assays, comet assays, and γ-H2AX upregulation data.Evidence: Figures 1C, 1E, 2F, 2G, 3B show statistically significant reductions in DNA repair and increased DNA damage with spike protein expression.
the SARS-CoV-2 spike protein significantly inhibits DNA damage repair
Abstractreviewer’s wording - supportedReviewer 2Spike protein localizes in the nucleus and impedes recruitment of BRCA1 and 53BP1.Subcellular fractionation and immunofluorescence data show nuclear localization and reduced foci formation of BRCA1 and 53BP1.Evidence: Figure 3A shows spike in nuclear fractions; Figures 3D-3G show reduced 53BP1 and BRCA1 foci.
“spike protein markedly inhibited both BRCA1 and 53BP1 foci formation”
Results - supportedReviewers 2, 3Spike protein impairs V(D)J recombination in vitro.The in vitro V(D)J reporter assay shows a significant reduction in GFP+RFP+ cells with spike overexpression.Evidence: Figure 4B-C show flow cytometry plots and quantification with p < 0.0001.
“spike protein overexpression inhibited RAG–mediated V(D)J recombination in this in vitro reporter system”
Figure 4 - supportedReviewer 3Spike protein inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site.The claim is supported by foci formation assays showing reduced BRCA1 and 53BP1 foci after γ-irradiation in spike-expressing cells.Evidence: Figure 3D-G show reduced 53BP1 and BRCA1 foci.
“We found that the spike protein markedly inhibited both BRCA1 and 53BP1 foci formation (D–G).”
Results
Data authenticity concerns
None foundAn adversarial read for patterns associated with data that may not be genuine: results that look too clean, implausibly large effects, duplicated data or images, and methods that do not match the results reported.
Checked — nothing surfaced.
Reporting gaps
4 findings · worst highRequired detail the manuscript never states — study design, biological variables, ethics approval and consent, key resources, statistical reporting, data and code availability, and overall transparency.
- Data and code not sharedAssessed
- Key resources not identifiedAssessed
- Statistical reporting gaps (tests, assumptions, effect sizes)Assessed
- Study design lacks key rigor safeguardsAssessed
The introduction cites clinical studies showing delayed adaptive immunity in COVID-19 and the interdependence of immune and DNA repair systems. The study objective to investigate whether SARS-CoV-2 proteins hijack the DNA repair system logically follows from the cited evidence. However, the paper does not explicitly discuss how it addresses limitations of prior work, such as the lack of mechanistic studies.
“Recent clinical studies have shown that SARS–CoV–2 infection extraordinarily affects lymphocyte number and function [,,,].”
“Therefore, we aimed to investigate whether SARS–CoV–2 proteins hijack the DNA damage repair system, thereby affecting adaptive immunity in vitro.”
“As two critical host surveillance systems, the immune and DNA repair systems are the primary systems that higher organisms rely on for defense against diverse threats and tissue homeostasis.”
The paper does not describe randomization of samples or blinding for most assays, lacks a priori power analysis, does not define inclusion/exclusion criteria or outlier handling, and does not report independent replication. Biological replicates are distinguished ('independently collected or prepared samples') and controls (empty vector) are used, but these are insufficient to meet the threshold for pass.
“All scoring was performed under blinded conditions.”
“All experiments were repeated at least three times using independently collected or prepared samples.”
“All scoring was performed under blinded conditions.”
“All experiments were repeated at least three times using independently collected or prepared samples.”
“Effect of empty vector (E.V) and SARS–CoV–2 proteins on NHEJ DNA repair.”
“All scoring was performed under blinded conditions.”
“All experiments were repeated at least three times using independently collected or prepared samples.”
The paper specifies HEK293T and HEK293 cells from ATCC and describes culture conditions (DMEM, 5% CO2, 37°C, FCS, antibiotics). Sex of the cell line is not reported, but this is not typically considered critical for cell line studies. Other variables like age and weight are not applicable.
“HEK293T and HEK293 cells obtained from the American Type Culture Collection (ATCC)”
The paper states that the study does not involve humans or animals, and the cell lines are commercially available. Therefore, no ethics approval is needed.
“Not applicable, because of this study not involving humans or animals.”
“Not applicable, because of this study not involving humans or animals.”
“Institutional Review Board Statement Not applicable, because of this study not involving humans or animals. Informed Consent Statement Not applicable, because of this study not involving humans.”
Antibodies are identified with vendor and catalog numbers but lack RRIDs, clone information, and validation data. Cell line authentication and mycoplasma testing are not reported. Software (GraphPad 8) is named but without version for Fiji. Plasmids are well-identified with Addgene numbers.
“Antibodies against His tag (Cat #12698), H2A (Cat #12349), H2A.X (Cat #7631), γ–H2A.X (Cat #2577), Ku80 (Cat # 2753), and Rad51(Cat #8875) were purchased from Cell Signaling Technology (Danvers, MA, USA).”
“pHPRT–DRGFP and pCBASceI were kindly gifted by Maria Jasin (Addgene plasmids #26476 and #26477) []. pimEJ5GFP was a gift from Jeremy Stark (Addgene plasmid #44026) [].”
“Antibodies against His tag (Cat #12698), H2A (Cat #12349), H2A.X (Cat #7631), γ–H2A.X (Cat #2577), Ku80 (Cat # 2753), and Rad51(Cat #8875) were purchased from Cell Signaling Technology”
“pHPRT–DRGFP and pCBASceI were kindly gifted by Maria Jasin (Addgene plasmids #26476 and #26477)”
“Data were analyzed by Student’s t test or ANOVA followed by Tukey’s multiple-comparison tests using GraphPad 8.”
The paper uses Student's t-test and ANOVA with Tukey's post-hoc, but reports p-values only as thresholds (e.g., p < 0.01). No effect sizes with confidence intervals are provided. Assumptions of normality and equal variance are not mentioned. Figures show bar plots with error bars but no individual data points. Statistical software (GraphPad 8) is identified.
“Data were analyzed by Student’s t test or ANOVA followed by Tukey’s multiple-comparison tests using GraphPad 8.”
“*** p < 0.001, **** p < 0.0001.”
“Data were analyzed by Student’s t test or ANOVA followed by Tukey’s multiple-comparison tests using GraphPad 8.”
“** p < 0.01, *** p < 0.001, **** p < 0.0001”
“Data were analyzed by Student’s t test or ANOVA followed by Tukey’s multiple-comparison tests using GraphPad 8.”
“** p < 0.01, *** p < 0.001, **** p < 0.0001.”
The paper states 'The data presented in this study are available in the main text and supplementary materials', which is not a concrete data availability statement. No repository deposit, accession numbers, or code sharing are provided. For a study generating experimental data, this is a significant transparency gap.
“The data presented in this study are available in the main text and .”
“The data presented in this study are available in the main text and .”
“Data Availability Statement The data presented in this study are available in the main text and .”
Methods are sufficiently detailed for replication. Limitations are discussed (in vitro system, lack of in vivo evidence). Conclusions are proportional to the in vitro data. Funding sources and conflicts of interest are declared. However, no reporting guideline (e.g., ARRIVE) is referenced.
“Although no evidence has been published that SARS–CoV–2 can infect thymocytes or bone marrow lymphoid cells, our in vitro V(D)J reporter assay shows that the spike protein intensely impeded V(D)J recombination.”
“Although no evidence has been published that SARS–CoV–2 can infect thymocytes or bone marrow lymphoid cells, our in vitro V(D)J reporter assay shows that the spike protein intensely impeded V(D)J recombination.”
“The authors have declared that no competing interests exist.”
Broken references and links
None foundReferences checked against Crossref, OpenAlex and Retraction Watch for retractions and resolvability, plus declared data and code links probed for whether they resolve to content matching the paper.
Checked — nothing surfaced.
Checked 29 references by DOI: 28 verified — 1 no DOI (shown, not verified).
- NO DOISARS-CoV-2 Spike protein promotes hyper-inflammatory response that can be ameliorated by Spike-antagonistic peptide and FDA-approved ER stress and MAP kinase inhibitors in vitroNo DOI in the reference — shown for manual review; not independently verifiable (not a fabrication signal).
1 data/code link checked; 1 live.
- datahttps://www.mdpi.com/article/10.3390/v13102056/s1LIVEHTTP 200Resolves, but the content could not be matched to the paper.
Copyediting
8 minorWording, consistency and formatting errors that need correcting before submission.
No major wording or formatting errors. 8 minor suggestions below.
8 copyedit issues flagged: mostly consistency, clarity, typo.
- MINORconsistencyFigure 2 legend“NS (Not Significant): * p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.”→ Remove the asterisk from the NS line and correct to 'NS (Not Significant): p > 0.05' to avoid confusion.The asterisk conventionally indicates significance; using it for non-significant results is misleading.
- MINORclarityFigure 2 legend, line 2“For H2O2: 100 µM, 1 h.”→ Use consistent formatting: 'H2O2' should be written as 'H2O2' with subscript 2.The paper uses H2O2 in the text and H2O2 in the legend; standardize.
- MINORtypoResults, section 3.1, first paragraph“and ()”→ Replace with 'and Figure 1A' or similar, as the parentheses are empty.Likely a formatting error where figure references were omitted.
- MINORconsistencyData Availability Statement“The data presented in this study are available in the main text and .”→ Complete the sentence, e.g., 'and in the supplementary materials.'The sentence appears truncated.
- MINORclarityFigure 1 legend“NS (Not Significant): * p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001”→ Remove 'NS (Not Significant): * p > 0.05' as it is contradictory; NS should not have asterisks.The notation is confusing; NS is typically not marked with a star.
- MINORconsistencyAbstract“SARS–CoV–2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro”→ Consider lowercasing 'Spike' to 'spike' for consistency with the rest of the text.The title uses 'Spike' capitalized, but the body text uses 'spike'.
- MINORpunctuationData Availability Statement“The data presented in this study are available in the main text and .”→ Complete the sentence: '... in the main text and supplementary materials.'The sentence ends abruptly after 'and'.
- MINORgrammarResults, section 3.1“Nsp1, Nsp5, Nsp9, Nsp13, Nsp14, and Nsp16 proteins are indeed localized in the nucleus, and nucleoproteins are mainly localized in the cytosol.”→ Consider rephrasing to avoid the comma splice: '... the nucleus, while nucleoproteins are mainly localized in the cytosol.'The sentence uses a comma to connect two independent clauses.
As published, the study is methodologically plausible and largely reproducible from the methods text, but readers should weigh the non-functional data-availability statement (no repository), the unreported cell-line authentication/mycoplasma status, and the fact that no reported statistic could be machine-verified. The Discussion's spike-vaccine side-effect claim and the comet-assay cell-count inconsistency ('20 fields × ~100 cells' vs 'n > 200 comets') are the most concrete points warranting a correction or author response; independent replication or formal data deposition would materially increase confidence in the findings.
- 1.HIGHdata codeComplete the truncated Data Availability statement ('...available in the main text and .') and deposit the underlying flow-cytometry, comet-assay image, and reporter-assay data in a public repository (e.g., Figshare, Zenodo) with a DOI, naming the supplementary file format.A data-driven paper whose only data-access statement is a broken sentence and which provides no repository is a concrete transparency defect that undermines reproducibility.
- 2.HIGHreportingTemper the Discussion claim that 'full-length spike-based vaccines may have side effects by inhibiting adaptive immunity' by explicitly stating that this is an untested extrapolation from in vitro data and that no in vivo vaccine evidence was produced.The independent claim audit rated this statement as overstated; over-claiming beyond the in vitro evidence is the most likely reviewer objection.
- 3.HIGHstatisticsReplace threshold-only p-values ('** p < 0.01', '**** p < 0.0001') with exact p-values and add effect sizes with 95% confidence intervals for the main spike-vs-E.V comparisons in the Figure 1–4 legends and Results text.Threshold-only p-values are imprecise reporting that cannot be verified or reproduced by readers, and no effect sizes/CIs are provided anywhere.
- 4.HIGHrigorAdd a statement in Materials and Methods 2.3 on cell-line authentication (STR profiling) and mycoplasma testing — or state explicitly that neither was performed — together with the reason.Cell-line identity and mycoplasma status are standard key-resource reporting items; their absence was flagged by all three reviewers.
- 5.HIGHstatisticsReport whether normality and equal-variance assumptions were checked for the t-tests/ANOVA in Methods 2.9, and state the fallback (Welch's t-test, non-parametric tests) if assumptions were violated.Assumptions are never verified despite parametric tests being used, which is a statistical-transparency gap.
- 6.MEDIUMrigorReconcile the comet-assay quantification inconsistency: Methods 2.6 states '20 fields with approximately 100 cells' (~2000 cells/sample) while the text reports 'n > 200 comets' — correct the numbers so they are consistent.An unresolvable internal contradiction about the analyzed sample size is the kind of detail an informed reader will doubt.
- 7.MEDIUMrigorAdd a sample-size justification (a priori power analysis based on expected effect sizes) for the reporter assays and V(D)J assay, or explicitly label the experiments as exploratory in Methods 2.9.No power analysis is reported, which limits the strength of the negative and positive claims.
- 8.MEDIUMrigorDescribe blinding for assays other than immunofluorescence scoring (e.g., flow-cytometry quantification) and state pre-specified inclusion/exclusion criteria and outlier-handling rules in the Methods.Only IF scoring is reported as blinded, and no data-handling rules are given, which is the dominant study-design gap.
- 9.MEDIUMcopyeditRepair the empty parenthetical 'and ()' figure reference in Results 3.1 and replace the '[,,,]' / '[,]' bracket citation placeholders in the Introduction with the actual reference numbers.Empty citation placeholders and an empty parenthetical are production errors that break traceability of the cited evidence.
- 10.MEDIUMcopyeditCorrect the contradictory significance notation in the Figure 1 and Figure 2 legends: 'NS (Not Significant): * p > 0.05' should read 'NS: p > 0.05' with no asterisk.Using an asterisk for a non-significant result is misleading and was flagged both by copyedit and the integrity check.
- 11.LOWcopyeditStandardize formatting: use 'H2O2' with subscript consistently in the Figure 2 legend, lowercase 'Spike' in the title to match the body text, and fix the comma splice in Results 3.1 ('...in the nucleus, and nucleoproteins...' → '...in the nucleus, while nucleoproteins...').Minor consistency issues that a professional copyedit would catch and that reduce polish.
- 12.LOWdata codeAdd RRIDs or clone information and working dilutions for the antibodies in Methods 2.1, and specify the Fiji version used in Methods 2.6.Completes the key-resource identification so other labs can reproduce the antibody conditions exactly.
- 13.LOWreportingState whether any data points were excluded and the rationale if so, and reference an applicable reporting guideline (e.g., ARRIVE or a cell-line reporting checklist) in the Methods.Closes the remaining transparency gaps flagged by reviewers; no guideline is currently cited.
The star rating is the report’s one-glance summary. Every paper starts at 5★ and loses stars for the concrete problems the review finds — so a rating is never a vague average, it’s a running total you can read line by line under “How this rating was calculated.”
- Reporting — 8 dimensionseach dimension that fully fails−½★
- each dimension partially met−¼★
- Statistics · Integrity · Claimseach serious problem−1★
- each medium problem−½★
- Citationseach retracted or unverifiable reference−¼★
- Copyeditonly when the manuscript needs a full edit−½★
The rating never drops below 1★, and a demonstrable critical failure (an impossible statistic, a proven ethics violation) caps it at 1★ on its own — so the stars can never look healthy when the verdict is CRITICAL.
The rating draws on a panel of agents. Three independent Kaimen Rigor reviewers grade the eight dimensions below across several independent passes (the shown verdict is their majority vote — steadier than any single run), isolate the paper’s major claims and check its own evidence backs them, and flag integrity concerns. Alongside them, a citation agent resolves every reference against Crossref, OpenAlex, and Retraction Watch; a statistics agent recomputes reported tests; and rule-based checks verify that declared data/code links actually resolve. Full text is required — an abstract-only submission is not analyzed.
Graded against NIH, MDAR, ARRIVE 2.0, CONSORT, EQUATOR, and RRID guidelines. A dimension that doesn’t apply to the study type is skipped, never penalized.
This Kaimen Rigor review is model-assisted and is not a substitute for formal expert review. It complements human evaluation by surfacing potential methodological concerns — verify each finding against the source.