Microbiome–Immune Interaction and Harnessing for Next-Generation Vaccines Against Highly Pathogenic Avian Influenza in Poultry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Highly pathogenic avian influenza have resulted in significant impacts on global poultry production, epizootics, even a persistent threat to public health since 2020. In this review, Sang et al. provided a comprehensive overview for the diversities of HPAI vaccine platforms, the advantage/limitation of each vaccine platform, along with the influences of microbiome-immune crosstalk vaccine efficacy. Besides, author also introduced the up-to-date AI-derived technology and applications in refinement of HPAI vaccines. Throughout the manuscript, authors gave detailed, robust, informative, and advanced summarizations, as well as proposed some challenges and research gaps for future investigations, to generate a novel, broadly-protective next generation HPAI vaccine, to mitigate/respond to zoonotic adaptation and pandemic emergence. It remains few points to be corrected:

1). Line 150, …HAPI invasion? It should be corrected into HPAI?

2). Line 168, …which are crucial for priming “ILF”-23-dependent B-cell activation. What ILF meant? Please clarify the word, it should be IL-23 or ILF-23? If ILF, please provide the full name but not abbreviation.

3). Line 313, what the DIVA-compatible construct meant? Same with Line 375? Please clarify it and corrected if needs.

Author Response

Please see point-by-point response in the attached file

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reviewed the recent advances of the microbiome-immune crosstalk, commensal vector platforms, and AI-enhanced vaccine development against highly pathogenic avian influenza, providing new insights and strategies for the development of next-generation influenza vaccines.

In the Figure1,   The presence of “Table 3.” in the figure causes some confusion, which was mentioned as “Table 1) in the caption. It is suggested to replace “Table 3” and “Table 1” with “c”, and indicate "a" and "b" in the caption.

Author Response

Please see in the point-by-point response in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review provides a comprehensive synthesis of emerging strategies at the intersection of microbiome research, vaccine development, and artificial intelligence (AI) applications for controlling highly pathogenic avian influenza (HPAI). The authors highlight the limitations of current vaccine platforms and make a compelling case for integrating microbiome-immune crosstalk into next-generation vaccine design. The focus on commensal bacteria as vectors, postbiotic/synbiotic adjuvants, and AI-driven optimization aligns with cutting-edge advancements in vaccinology, and the emphasis on One Health integration adds significant relevance to addressing zoonotic risks.

To strengthen the manuscript, the following revisions are recommended:

1. Clarify species-specific microbiome variations: While the authors note differences between chicken (Firmicutes-dominated) and duck (Proteobacteria-rich) microbiomes, more detail on how these differences directly impact vaccine formulation would be valuable. For example, specifying adjuvant dosages or vector modifications tailored to ducks could enhance the practical applicability of the review.
2. Expand on translational challenges: The section on challenges (e.g., ecological safety, scalable manufacturing) is somewhat brief. Expanding on real-world examples—such as case studies of biocontainment failures or scaling bottlenecks in pilot projects—would provide deeper context for researchers and policymakers.

Validate AI tool applications: The claims about AI-driven antigen design and early detection of viral drift would benefit from referencing specific validated models or recent trials. For instance, citing performance metrics (e.g., accuracy rates of EVEscape in forecasting HPAI mutations) would reinforce their reliability.

3. Strengthen One Health integration: While the One Health framework is introduced, concrete examples of interdisciplinary collaborations (e.g., wildlife surveillance paired with poultry vaccine deployment) would illustrate how this approach mitigates zoonotic risks more tangibly.​
4. Address formatting inconsistencies:

1) Tables 1 and 2 contain minor formatting issues (e.g., inconsistent bullet points, truncated headers) that should be standardized for clarity.

Here are the technical corrections presented as formal peer-review comments, with verified line numbers based on the provided PDF content:

2) Line 48: The term "SOFA" is used as an acronym for short-chain fatty acids. This should be corrected to the standard abbreviation "SCFA" throughout the manuscript. 

3) Line 150: "HAPI invasion" appears to be a typographical error. This should be revised to "HPAI invasion" for consistency with the manuscript's standard terminology. 

4) Line 264:  The phrase "We have recent performed meta-analyses" contains a tense error. Correct to "We recently performed".

5) The reference citation format is disordered and needs to be standardized.

6) Line 313 375: The acronym "DIVA" is used without prior definition in line 313.

Author Response

Please see in the point-by-point response in the attached file.

Author Response File: Author Response.pdf