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Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper

• The author is trying to develop a universal vaccine against Influenza. Their use of equine H3N8 hemagglutinin as a vaccine immunogen is a promising and innovative strategy. They demonstrated the equine H3N8 vaccine induces broad protection against multiple influenza strains. Also, it effectively protects both mice and ferrets from influenza infection. It can induce neutralizing antibodies and provide heterosubtypic immunity against various strains of influenza A viruses in animal models. 

Major comments  - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• The study's strength lies in the detailed experimental design used and the use of different animal models. The vaccine’s potential also showed significant cross-reactivity and neutralization effects on various strains of influenza. However, the limited sample sizes used in some of the experiments and the possibility of generalizing the findings across different populations should be looked into.

• Identifying conserved epitopes on the HA protein that are less prone to mutations could be a focus of molecular studies.

• A molecular analysis of the mechanisms of viral evolution and immune evasion could inform vaccine design strategies.

Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• The specific assays used in measuring the antibody responses may need detailed explanations to provide a better understanding. Additional detailed legends to describe some significant observed patterns in the Figures illustrating antibody responses may also be included. There is also a need to address some inconsistencies in the use of some terms related to antibody types (e.g., bnAbs vs. nAbs) throughout the article to avoid confusion.

Comments on reporting - information on the statistical analyses or availability of data.

• The authors wrote a few times “data not shown” (e.g. line 147, 240, 306, 359, 457). However, it is hard to convince readers without showing raw data. Maybe they could include the data in supplementary files or simply remove those points instead. If there is a real need to include those points but the authors could not prove the raw data, they might discuss those data in the discussion section instead.

• The authors may provide more details on the statistical methods used especially with specific tests used for comparisons and criteria for significance levels (i.e., p-values).

Suggestions for future studies

• For future studies, the long-term efficacy of the immunity conferred by the H3N8 vaccine in both animal models and human trials may be investigated. Also, further studies with focus on cellular immune responses together with humoral responses can be explored.

Conflicts of interest of reviewers

• There are no conflicts of interest to declare.

Inline commenting section

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• Introduction paragraph 2. It said “Due to the limited efficacy of current…”

  • The authors have stated the importance of targeting conserved antigenic sites on the (HA) protein for broad protection, a deeper molecular analysis of these epitopes and their interaction with the immune system could be explored

• Introduction paragraph 2. It said “A universal influenza vaccine capable of providing broad protection against group 1 and group 2 influenza viruses,…” 

  • what does it mean by group 1/2 ?

• Introduction paragraph 3. “Neutralizing antibodies (nAbs) generally bind to epitopes”

  • Is this broadly neutralizing antiody?

• Introduction paragraph 3. “Humans also recognize antigenic sites …”

  • Could the authors clarify this sentence? How do human recognize antigenic sites?

• Introduction paragraph 3. “Whether or not enhanced disease … avoid stem-binding Abs ”

  • These two sentences are not connected well. Could the authors revise them?

• Introduction paragraph 4. “ However, bnAbs can occur … infections.”

  • Is it bnAbs ?

• Introduction paragraph 5. “We recently discovered an alternative immunogen…  group 1 and 2 viruses”

  • The authors may need reference to support this statement. Although it is rare, broadly neutralizing antibodies are not common solely for H3N8. It would be beneficial to justify this perspective as well.

• Result paragraph 1. “To assess for the effect of prior influenza exposure … and did not detect reactivity (data not shown).

  • The authors may compare the equine H3N8-induced antibodies with well-characterized broadly neutralizing antibodies to identify similarities and differences.

• Result section 2, paragraph 2: “In further agreement ….  influenza strains (Fig 2C)”

  • The authors may also investigate the role of T cell responses in addition to antibody responses in mediating protection

• Result section “Vaccination protected mice from heterosubtypic challenge”

  • The authors may also perform direct comparisons of the equine H3N8 vaccine with other candidates in terms of immunogenicity, breadth of protection, and safety...this could provide more novel features

Competing interests

The authors declare that they have no competing interests.