Review 2: "Evolution of Omicron Lineage Towards Increased Fitness in the Upper Respiratory Tract in the Absence of Severe Lung Pathology"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: This study led by Drs. Wit and Munster performed a comprehensive comparison study among pre-dominant (XBB, BQ and EG.5.1, etc.) and recent circulating Omicron variants (JN.1) regarding their virological properties including fusogenicity, pathogenicity, fitness and transmissibility in primary cell models and Syrian hamster model. They concluded that 1) Omicron variants showed increased entry efficiency compared to ancestral WA-1. 2) JN.1 exhibited further attenuated pathogenesis in the lower respiratory tract in hamsters either I.N. or I.T. infection routes. In addition, the results from transmission studies demonstrated that JN.1 did not transmit between hamsters in neither contact nor non-contact settings. 3) In the primary cell studies, they used nasal epithelial and ihLOs to recapitulate URT and LRT infection, respectively. The results indicated that Omicron variants displayed replication advantages in nasal epithelial cells as that of Delta while Omicron variant expect XBB.1.16 failed to replicate in ihLOs, which were consistent as in vivo results. 4) Lastly, they demonstrated JN.1 exerted distinct genetic divergence compared to pre-existing Omicron variants and XBB.1.16 infection triggered robust ISGs among other studied variants.

Below comments shall be considered to further improve the manuscript:

1. Line 146, intratracheal inoculation is not physiological relevant thus considerably compromise the conclusion in this paragraph. 

2. The binding affinities such as KD between the studied spikes and ACE2 should be measured to directly show the difference instead of a pseudovirus entry experiment. In addition, the spike expression level in different pseudotyped viruses might not be identical; did the author normalize the activities of pseudosviruses to the equal level when comparing their entry efficiency (Fig. S1).

3. The authors demonstrate that various Omicron variants replicate more efficiently than Delta in nasal epithelium cells (Fig. 3) which provide evidence that Omicron could outcompeted the predominant strains and mainly cause URT infection in humans. However, the authors demonstrate that JN.1 exhibited less replication in nasal epithelial cells than predominant Omicron variants such as EG.5.1, which is not representative to the real-world situations. The same scenarios can be seen in the in vivo pathogenesis and transmission experiments in hamsters. The authors should give an explanation for this paradox.

4. The authors demonstrate the distinct antigenicity of JN.1 among other variants, which may provide insight into the domination of JN,1. The authors should show the neutralizing activities of JN.1 against pre-existing immunity to demonstrate its immune evasion abilities, which may provide the potential mechanisms for replacing pre-existing variants.

5. The authors should give a comprehensive discussion regarding the evolutionary trend of Omicron lineages based on their data and discuss the potential key factors that drive such evolution. On the other hand, the author should also correlate the enhanced ISGs expression and evolution trend in Omicron emergences. Besides, the authors should give explanations that why hamsters may not be the relevant animal model to recapitulate the Omicron JN.1 infection. 

Some minor points:

1. In line 228, 9 out of 10 hamsters were infected by donors in mRNA level on Day 3. Similarly, 8 out of 10 were positive in terms of EG.5.1 infection. Are they typos or other criteria were used to determine positive.

2. In line 310, capitulate or recapitulate?