Review 1: "Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce superior immune responses than single dose vaccine regimens in mice"

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.

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Review:

• The study utilizes both inbred and outbred mice to characterize the immune responses generated from homogenous vs. heterologous vaccination strategy against SARS-CoV-2. Outbred mice might more closely resemble the genetically diverse human population and consequently the variability of vaccine-elicited immune responses. However, the study didn’t include naïve control mouse groups. That is an important control to define the baseline T-cell responses.

• A challenging study to evaluate the efficacy of the heterologous vaccine regimen was not attempted to truly evaluate vaccine efficacy. Although we understand how this might be outside the scope of the study, we still think this point needs to be discussed and highlighted in the manuscript.

• The authors highlighted that the heterologous vaccine group elicited better antibody responses (line 143) but according to figure 2D the homologous RNA vaccine group had the highest IC50 neutralizing titers even higher than the heterologous vaccinations. The nAb responses are biologically relevant and are critical for limiting viral replication.

• Although nAb responses were not significantly different, the heterologous vaccine-elicited more robust and significant T-cell responses than homologous vaccines substantiating the authors’ claims

• CD8+ T-cells secreting type-2 cytokines like IL-4 and IL-13 (Tc2) can exacerbate respiratory viral infections as seen with RSV. Vaccine-induced Tc2 responses were not examined in this study. This point needs to be examined or at least discussed in the manuscript.