RR:C19 Evidence Scale rating by reviewer:
Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.
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Review: The manuscript (https://doi.org/10.1101/2024.06.13.598902) is well written, and the experiments are very nicely conducted. Overall, this study supports an evolutionary trend of selection pressure in the Omicron lineage towards adaptation in the upper respiratory tract. However, I have listed my concerns and suggestions to improve the manuscript.
Major Concerns:
Previous reports by PMID:37221221, and other groups (PMID:37221221, PMID:38812550, PMID:32893735, PMID:36108630, PMID:36959194) have carried out similar studies. So, I am concerned about the novelty of this study.
What is the cause of attenuation of viral replication? Is it due to the Furin cleavage site (PRRAR) in the spike protein of all the Omicron variants used in this study? Because viruses are propagated on VeroE6 cells, which results in the disruption of the furin cleavage motif due to virus adaptation to cells that express TMPRSS2, where the virus is forced to enter by the endosomal route and adapt the spike to cathepsins (see PMID 35019723). It is widely known that this results in the in vivo attenuation of SARS COV-2 (see PMID 33494095) and that virus propagation on cells expressing TMPRSS2 restores virulence (PMID: 34960703).
It is important to show that the changes observed in animal models and in-vitro human epithelial culture models upon infection with Omicron variants are correlated with the change observed in human patients or patient data available in the literature. In my view, this study will gain much strength if the author could show the gene expression analysis or proteomics analysis of blood plasma and correlate with the published results of human patients infected with Omicron variants.
Did the authors find any differences of virus specific antibody and T-cell responses in these models? Optimal adaptive immune response requires intact IFN-I pathway.
It would be better if the authors could provide Highlights and Graphical Abstract.
Did the authors check the cytokine profile upon infection with Omicron variants? Explain.
Minor Concerns:
Abstract is short.
The study reveals only characterization and doesn’t have any mechanistic approach. Justify.
Figure1A/B: The data distribution looks very dicey. All data points are almost similar. Verify.
Figure 2F,4I: The IHC score should be labelled with Viral antigen used.
Figure3A/S3A: There are no error bars in most of the datapoints.
Figure 5A: How can the authors negate the notion that Omicron variants, BA.1,XBB.1.5,EG.5.1,JN.1 doesn’t infect iPSC derived ihLOs.
Figure 7B: What is the relative expression profile for type I IFN response (IFN-Alpha/Beta)?
Figure S3A: In EG.5.1, 72and96 hours data point is not significant which is strange? Which statistical test, verify.
Figure S4C: Distorted lung images can be replaced with fresh one.
Figure S5A: Authors claiming the lamellar bodies in TEM images is doubtful. Verify and replace the image.
Figure S5B: The p-value is written, but upon comparison with whom. Make it clear in legends.
Figure S5C: LDH levels in XBB.1.5 at 0hr seems to be high for both groups. Rectify.
Put name of statistical test in all figure legends.
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