Review 2: "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability"

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

In the manuscript entitled "A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability", by Voigt et al. the authors propose an NLC formulated self-amplifying mRNA against SARS-CoV-2 that is thermostable. Of note, the main study claims are generally justified by its methods and data. However, there are some limitations and issues that need to be addressed:

Technical points 

1. Instability is the main drawback in running RNA constructs on agarose gel due to heat generation during electrophoresis. As a result, RNA degradation and smear formation may be observed. You mentioned that you had run RNA for 45 min at 120V (which is a relatively high voltage). Please double-check if you have done the electrophoresis at 4 C or lower voltage and edit the protocol accordingly. 

2. In the results and discussion sections, please mention the yield of the obtained saRNA using the suggested in vitro transcription protocol and following purification and encapsulation steps, as it is critical for supporting your claims regarding the potential of the introduced platform for large-scale production.

3. The ability of a vaccine candidate in pathogen clearance is of importance. It is not recommended to jump into clinical trials without such evidence as it is mentioned in paragraph 1 of the discussion section. Consequently, challenge study with the SARS-COV2 in the lung of the immunized mice and viral clearance is crucial in such studies as neutralizing antibody assays are necessary but not sufficient.

Comparison with current vaccines

4. Thermo-stability and cost-effectiveness are both important in the context of providing vaccines for low-income countries. Although the designed vaccine candidate is stable at room temperature for a long time, nano-lipid encapsulated vaccines are more expensive than other platforms such as attenuated or inactivated vaccines. Accordingly, many African, Asian, and South American regions of the world can not afford even one dose of it, let alone several doses, as it is recommended for preventing different variants of COVID-19. I recommend reconsidering this claim in the introduction section based on this fact.

5. In order to conclude the advantages of the designed vaccine over existing licensed vaccines such as Moderna or Pfizer-BioNTech, they needed to be used as control groups in the study. Above mentioned vaccines have undergone clinical trials, tested on billions of people, and shown to be efficient. Accordingly, you need more robust and comprehensive pieces of evidence to suggest the designed vaccine as an alternative to them. Please avoid insisting on its superiority throughout the discussion section unless you perform further stability and comparative immunogenicity studies. Some statements in the discussion section should be rewritten accordingly.