Review 2: "A live attenuated vaccine offers superior mucosal and systemic immunity to SARS-CoV-2"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

***************************************

Review:

The article entitled, “A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants,” describes the challenge and analysis of hamsters with control vaccines as well as the novel vaccine candidate sCPD9. This study suggests the vaccine candidate offers greater protection in many regards as compared to mRNA and adenovirus-based vaccines. This study is well done, well-written and is important in the fact of immune-evading SARS-COV-2 variants. Overall, the claims are generally supported by the data and methods used, although I had some lingering questions regarding the significance of certain aspects of the analysis as described below. Decision-makers should consider the claims in this study actionable with limitations based on the methods and data.

1. The introduction could be improved by including information on LAVs related to COVID rather than a broad discussion of LAVs in general and being more thorough in general (Lines 29-38).

2. Why were the animals challenged with Delta-only rather than Omicron SARS-CoV-2 variants? This lessens the impact of the experiment in the current environment.

3. Overall, the small group numbers decrease the significance of the findings. I also question the effects of the difference in inoculation (sCPD9 is IN and others were IM as well as a difference in doses). Did you have a control for these factors? This difference could explain some of the findings, particularly some of the differences in activated cell groups as well as the histopathological results.

4. Please describe the single cell methods in greater detail. Did you utilize the Mesocriceous auatus reference genome as a control in all of your analysis? This genome is not a high quality genome and would likely affect the results.

5. In line 378-379 you suggest significant differences occur in figures 1F and 1G, however, it appears the significant differences only occur on 2DPC and not 5DPC?

6. Please include a description of how lungs were scored (referenced in line 391)

7. Lines 440-444 discuss the serum neutralization of various SARS-CoV-2 variants. In line 442-443 it suggests the variants are neutralized at early time points which is not apparent in the Omicron figure as at 0DPC there is no observed difference in neutralization. Please modify these sentences to fit the findings. In addition, the threshold lines on these figures are not explained. Are they neutralization thresholds? If so, it does not appear the omicron variant is consistently neutralized with any of the treatments at early time points.

8. Line 456 suggests the Ad2-spike was less efficient at enhancing antibody titers but there are no significant differences at 5 DPC. Please explain and rephrase this statement.

9. Line 540-541 sentence describing higher frequencies of cells in lungs. If not significant, the sentence should be removed.

10. Line 554, Figure 4I, was significance calculated on these data points/or on this figure? If not, please remove/rephrase sentence.

11. The staining pictures in Figure 5 are beautiful. Great job!