Cellular Immunological Memory T Cells and IL15RA Gene Polymorphism in COVID-19 Vaccinated Individuals from Southern Brazil

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

In the revised manuscript, the authors address most of my questions. I have no further questions.

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have made significant improvements in the revised version of the manuscript. I agree with the publication of the manuscript in its current form.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript try to study SARS-CoV-2-specific T-cell immunological memory in individuals who underwent different vaccination regimens and possible relation to rs2228059 polymorphism in the IL15RA gene. The study is interesting since it may provide valuable insights into the state of immunological memory and the potential genetic factors influencing its persistence within this population. However, I noted that two different cohorts were used for the study. one involved 54 people for T-cell immunological memory and 443 people for the genotyping study (Table 4). Therefore, no conclusion could be obtained to know the relationship between the T cell memory and the genotypes. Therefore, the experimental design needs improving. Furthermore, in view of the limited people (54 total) in each group used in the study, the number included in the study is too small to obtain the reliable results. Another thing is on the ethical consideration, the author should mention clearly if the informed consent include determining rs2228059 polymorphism in the IL15RA genes of the participants.

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors report in this manuscript the immune memory cell population after COVID vaccination, with a focus on memory T cells and IL15RA gene. Overall, this study has merits and I support its publication after addressing the following questions:

 

1. The authors use CD45RA and CD27 staining to identify effector and central memory T cells. Is there a reason why the more standard marker pair CD44 and CD62L was not used?
2. All the gating and representative flow images should be replaced with publishable figures processed in software like Flowjo.
3. The authors report that no difference in rs2228059 polymorphism was observed between their participants and the HCPA Biobank. They should discuss more on what does this result imply. Does that mean the significant gene related to immune memory was not changed after COVID vaccine?

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

While the study provides valuable insights, the preliminary nature of the findings is evident.

ABSTRACT

- The phrase “statistically significant for total T lymphocytes CD3+, as well as CD4+, CD8+ subsets” is imprecise. Specify which comparisons were statistically significant and provide exact p-values or effect sizes.

- Briefly mention the assay platform (e.g., flow cytometry, ELISpot) and the general approach to stimulation, to contextualize the findings.

- The novelty and significance of the findings are not well-articulated.

INTRODUCTION

- Minimize global epidemiological details and focus on the gaps in immunological memory research specific to heterologous vaccination. Also focus in the genetic determinants of immunological memory.
- While the IL15RA polymorphism is introduced, there is minimal discussion about its functional relevance beyond a general statement on binding affinity. No previous studies on its clinical significance or impact on vaccine responses are cited. Strengthen the rationale for including IL15RA rs2228059 by citing studies that link it to functional outcomes, immune cell regulation, or infection susceptibility. Associations between IL15RA polymorphisms and immune-related phenotypes may exist, but unless supported by direct experimental data, mechanistic claims about "differences in binding affinity" should be avoided.
- Consider ending the introduction with a precise hypothesis or set of objectives framed as testable questions. Rephrase the final paragraph to succinctly present the study objectives, saving methodological specifics for the Methods section.

METHODS
- What were the inclusion and exclusion criteria for participant recruitment? Were any demographic or clinical factors (e.g., age, sex, comorbidities, prior confirmed infection) used to stratify or exclude participants? How was prior SARS-CoV-2 infection status confirmed, solely via self-report, serological testing, or medical records?

- Clarify whether sample size calculations were conducted, and discuss limitations arising from the small cohort, especially for Group 3. Was a power calculation performed to determine sample adequacy for statistical analyses?

- Indicate the recruitment period.

- How long after the last vaccine dose or SARS-CoV-2 infection were the samples collected? Was the timing standardized across participants? Please, clarify the temporal relationship between vaccination/infection and sample collection.

-  Clarify the criteria for defining a "low response" to PHA-M and specify how many samples were excluded based on this criterion.

- Were all samples processed and cultured under identical conditions, or were there potential batch effects? How were these controlled for or accounted for in the analysis?

- Summarize the main adaptations in the main text and justify them, as EuroFlow protocols are highly standardized. Deviations must be clearly described and validated.

- Clarify the full set of controls used.

- Was the extracted DNA normalized for purity and concentration before genotyping?

- An independent cohort from the HCPA Biobank was used to estimate polymorphism frequency.

- What are the demographic characteristics (ancestry) of the HCPA Biobank cohort? How do these characteristics compare with the primary study cohort?

- Were genotype distributions tested for Hardy-Weinberg equilibrium (HWE) in both the study and biobank cohorts? Were minor allele frequencies (MAF) and call rates reported? Were any samples excluded due to poor amplification or ambiguous calls?

- Given that allele frequencies can vary by ancestry, were any steps taken to control for population stratification in genotype-phenotype analyses? Were ancestry-informative markers (AIMs) genotyped or were self-reported ancestries recorded?

- Consider reporting effect sizes and their confidence intervals for all key analyses.

RESULTS

- Justify the choice of the 14,000-cell threshold, possibly referencing technical requirements or quality control standards.
- Please include the results of the statistical comparisons between groups for all relevant demographic and clinical variables in Table 1.
- Consider clarifying how the absence of COVID-like symptoms was assessed (e.g., structured questionnaire, clinical evaluation).
- Provide measures of dispersion in the text (e.g., SD, IQR) for time since vaccination.
- The text mentions no significant differences among vaccination groups but does not provide detailed statistics or effect sizes. Include more detailed comparative statistics for T cell subsets by vaccination group.
- Authors should clarify and justify the inclusion and combination of the two distinct cohorts in the genetic analysis. Provide demographic and clinical summaries separately for each group to assess representativeness and potential confounders. Was statistical power calculated for detecting genotype associations, especially considering the stated small sample size?  Are exact p-values, effect sizes, or confidence intervals available for these comparisons? These parameters should be shown in Table 4.

DISCUSSION

- The discussion mentions using a principle similar to AIM assay but with direct flow cytometry evaluation using Euroflow markers and autologous serum to reduce nonspecific activation. Can the authors clarify the validation and reproducibility of their adapted assay compared to classical AIM assays? Was the peptide pool concentration optimized to avoid understimulation? How do they justify the concentration used given possible underestimation?

- Strongly emphasize the preliminary nature of these findings and avoid overstating conclusions.

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf