Review 1: "Rationally Designed Multimeric Nanovaccines using Icosahedral DNA Origami for Molecularly Controlled Display of SARS-CoV-2 Receptor Binding Domain"

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: The manuscript summarizes using icosahedral DNA origami (ICO) as a display particle for SARS-CoV-2 RBD nano vaccines, thereby improving the humoral and cellular responses obtained upon vaccination. The finding of this manuscript is of considerable interest and will contribute to the continued efforts in improving the effectiveness of SARS-CoV-2 Vaccines. However, The manuscript can be improved in certain aspects highlighted below:

1. The authors have used Female BALB/c mice. Biological variation is not considered. The authors have not provided any reasoning for using only female mice in the study. This might impose a bias in the study.

2. Viral Challenge with SARS-CoV-2 was not done in the present study. Challenge with live SARS-CoV-2 is often needed to assess the vaccine's efficacy. Although the authors have included immunogenicity data, such as binding and neutralizing antibody titers, the durability and effectiveness of these titers can be better assessed during a live viral challenge.

3. The authors have assessed IgG antibody responses obtained upon vaccination. Did the authors evaluate other antibody types, such as IgM, IgA, and IgG subtypes? It has been shown that both IgG and IgA play essential roles in neutralizing SARS-CoV-2.