Review 2: "Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, Against Both SARS-CoV-2 and Influenza A Viruses"

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: In this study, Ouyang et al. investigate the immunogenicity of an orally delivered recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine for SARS-CoV-2 and influenza viruses. The authors demonstrated that prime and boost immunization with this vaccine elicits strong antibody responses against SARS-CoV-2 and influenza viruses. The observed anti-influenza M2e antibody response was also consistent with in vivo protection in a mouse model of influenza H1N1 virus infection. This work demonstrated that oral immunization of an rVSV bivalent vaccine is a viable strategy to simultaneously induce systemic antibody immunity against two different respiratory viral pathogens.

This work builds upon a previous publication from the same group showing the same rVSV vaccine elicits strong systemic antibody responses against SARS-CoV-2 and influenza viruses when delivered intramuscularly (IM) or intranasally (IN). The authors did a great job citing relevant literature as well as discussing limitations of the study. The work is well-executed, well written, and will be of interest to researchers in the field of mucosal vaccines, recombinant viral technologies, and respiratory viruses. Overall, 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. See below some additional questions and comments that I have for the manuscript:

1. The authors provided endpoint titers for anti-SARS-CoV-2 IgG, but only provided the OD values at one selected dilution for IgA. What was the rationale behind that?

2. The observation that antibody responses against M2e induced by oral immunization was weaker than that of intranasal immunization is intriguing. Do the authors have any explanation for that?