Longitudinal transcriptional changes reveal genes from the natural killer cell-mediated cytotoxicity pathway as critical players underlying COVID-19 progression

Reviewer #1 (Public Review):

Summary:
Medina et al, 2023 investigated the peripheral blood transcriptional responses in patients with diversifying disease outcomes. The authors characterized the blood transcriptome of four non-hospitalized individuals presenting mild disease and four patients hospitalized with severe disease. These individuals were observed longitudinally at three time points (0-, 7-, and 28-days post recruitment), and distinct transcriptional responses were observed between severe hospitalized patients and mild non-hospitalized individuals, especially during 0- and 7-day collection time points. Particularly, the authors found that increased expression of genes associated with NK cell cytotoxicity is associated with mild outcomes. Additional co-regulated gene network analyses positively correlate T cell activity with mild disease and neutrophil degranulation with severe disease.

Strengths:
The longitudinal measurements in individual participants at consistent collection intervals can offer an added dimension to the dataset that involves temporal trajectories of genes associated with disease outcomes and is a key strength of the study. The use of co-expressed gene networks specific to the cohort to complement enrichment results obtained from pre-determined genesets can offer valuable insights into new associations/networks associated with disease progression and warrants further analyses on the biological functions enriched within these co-expressed network modules.

Weaknesses:
There is a large difference in terms of infection timeline (onset of symptom to recruitment) between mild and severe patient cohorts. As immune responses during early infection can be highly dynamic, the differences in infection timeline may contribute to differences in transcriptional signatures. The study is also limited by a small cohort size.

Reviewer #2 (Public Review):

In their manuscript, Medina and colleagues investigate transcriptional differences between mild and severe SARS-CoV-2 infections. Their analyses are very comprehensive incorporating a multitude of bioinformatics tools ranging from PCA plots, GSEA and DEG analysis, protein-protein interaction network, and weighted correlation network analyses. They conclude that in mild COVID-19 infection NK cell functionality is compromised and this is connected to cytokine interactions and Th1/Th2 cell differentiation pathways cross-talk, bridging the innate and the adaptive arms of the immune system.

The authors successfully recruited participants with both mild and severe COVID-19 between November 2020 to May 2021. The analyzed cohort is gender and acceptably age-matched and the results reported are promising. Signatures associated with NK cell cytotoxicity in mild and neutrophil functions in the severe group during acute infection are the chief findings reported in this manuscript.

Reviewer #3 (Public Review):

Summary:
Medina and colleagues explored transcriptional kinetics during SARS-CoV-2 between non-hospitalized and hospitalized cohorts and identified that early NK signaling may be responsible for less severe disease.

Strengths:
The paper includes extremely detailed analyses and makes an interesting attempt to link innate and adaptive responses. The analyses are appropriate for the data and described in clear language. The inclusion of late time points is interesting and potentially relevant to long COVID studies. Most findings were compatible with other detailed immune mapping during severe COVID-19.

Weaknesses:
1. The authors claim to be looking at the earliest stages of infection but this is not true as all patients enrolled are already symptomatic. The time points selected are unlikely to be useful clinically for biomarker selection as they are too late, and are likely beyond the point when the immune responses between severe and mild infection start to diverge.
2. The comparator timepoints between mild and severe cases do not match. The most comparison would be between day 7 of mild versus day 0 of severe which is already fairly late during infection.
3. The authors mention viral clearance but I see no evidence of viral loads measured in these individuals.
4. The cohort is quite small to draw definitive conclusions.
5. It is uncertain whether the results are applicable to current conditions as most infected people are immune experienced.
6. I found the discussion to be a bit too detailed and dense. I would suggest editing to make it more streamlined.