Review 1: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

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.

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

Review:

The long-term storage of RNA vaccines without freezing at -80°C is very challenging. The present study shows the long-term retention of a lyophilized RNA vaccine immunogenicity after storage at refrigerated and ambient temperatures. Although a similar article was published recently with similar conclusions (Muramatsu et al, Mol Ther, Feb 2022), the present study offers the advantage to investigate self-amplifying RNA (rather than mRNA) encoding SARS-CoV2 antigen (rather than influenza virus antigen). In my opinion, the current work merits being published as the results contain valuable information and constitute a major step forward in the current context of the COVID-19 pandemic.

As a general remark, I think that the experimental work is impressive, the text reads well, and the conclusions are adequate; however, the presentation of the data would benefit from additional work. For example, many results show comparison after prime and boost immunization; I think that it would be more robust if individual mice were represented by individual symbols (in Figure 2A for example). The statistical values are randomly displayed in the figures (although this is obvious, it would make figure 2B better if significance was shown between saRNA and control vector). Finally, very few experimental details are provided in the Figure legends—more details are needed.

More specific comments:

• Figures 2A and 2B: x-axis is not consistently labeled ("dose" in 2A, "post-immunization" in 2b) although the same parameter is considered in both graphs.

• Figure 2C: it is unclear whether the values were obtained after one or two doses of vaccines.

• Figure 3A: I do not see any reason why the order differs from panels B-D. Also, no statistics are depicted here, despite the results being significant. 

• Figure 3B-D: I think that results obtained after the prime immunization are distractive and should be moved to supplementary data. Instead, I think that the authors should merge the results obtained with the 3 different viral strains in one graph to highlight the differences in terms of neutralization (which is the take-home message here). Again, statistics values should be added (even if the results are not significant). 

• Figure 5. To my mind, percentages do not always reflect the response. The authors need to ascertain that the overall CD4 and CD8 T cell numbers remain comparable between vaccinated groups. The absolute numbers of cytokine-producing CD4 and CD8 T cells must be shown as well.

• Figure S3 and 6: since this section is the critical point of the paper, I would find it logical to include Figure S3 as the main Figure. Moreover, it would be more convincing to bring up dot plots/contour plots to reinforce the histograms.

• Line: 127-129: "All vaccine candidates were well-tolerated based on mouse general appearance and behavior, with no signs of weight loss or injection site reactogenicity, similar to previous studies with this saRNA/NLC platform." A supplementary figure supporting this statement should be added.

• Line 251: the authors should provide a short explanation of why the comparison was not possible.