Evaluation of a Bivalent Hexon-L1 and Fiber-2 Subunit Vaccine Candidate Against Homologous Fowl Adenovirus Serotype 4 Challenge in Chickens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “A Bivalent Subunit Vaccine Candidate Against Fowl Adenovirus Serotype 4” by Chu Xiaoran and coauthors formulated a vaccine candidate against FAdV-4 by producing the fiber and hexon proteins. Through molecular, serological, and experimental infection assays, they demonstrated an effective reduction in pathology after two doses of immunization in the groups. Overall, the study is interesting and presents a valid option to consider for the control of MSM in chickens. However, it is important to review some considerations in the manuscript.

Major Observations:
•    The authors state in their justification that current commercial vaccines offer limited protection. While this statement may be valid, the authors provided no evidence that their vaccine formulation is even comparable (if not worse), which is a significant limitation.
•    The authors conceptually overestimate its application to FAdV-4 (from the title), when they only performed the challenge with the same strain that gave rise to the vaccine.
Other points:
•    Given the scope of the study, the authors should provide a pathological description of HHS, differentiating, if possible, the more and less virulent forms of FAdV presentation.
•    Line 44: The term “molecular genotyping" is more appropriate.
•    Lines 75-78: This epidemiological support must also come from other studies already published.
•    Line 113: Also add the reference of the study where the virulence level of the strain used is evaluated and defined.
•    Section 2.10.1: The authors should provide more details about the challenge conditions and animal husbandry. Were the animals kept in isolators or single cages? Was any risk of contact between groups excluded? What were the rearing conditions (temperature, humidity, light, etc.)? It is important to detail these considerations to rule out any environmental factors influencing the immune response.
•    Section 2.10.2: The authors certainly included the main target organs (liver, heart associated with FAdV-4). However, why were intestinal segments not included? There is undoubtedly a greater likelihood of detecting lesions there than in the brain (which was included in the study).
•    Figures S1, S2, S3: Bands around 100 bp can be observed in the gels, including positive and negative controls. How did the authors rule out that this is an amplification byproduct or contamination?
•    Figure S8: In the "purified" hexon-L1 protein product, a smaller additional band can be observed. What is the reason for this? It should be described.
•    Table 5: Why was virus shedding not evaluated in the control group at all dpi? This is necessary to exclude a "natural" decay under the experimental conditions of the study that may have also affected the other 2 groups.
•    Discussions: There are entire sentences and paragraphs where the authors repeat results (i.e. lines 474-478; 499-450). This should be avoided and rewritten in discussion format.
•    Lines 501-503: The supporting references for this argument are not relevant (Salmonella?). This type of unrelated argumentative support occurs in other lines of reference throughout the manuscript and should be corrected.
•    Lines 504-505: The authors should avoid this type of claim here and throughout the manuscript. The presented study evaluated the vaccine candidate against the same strain from which the recombinant proteins originated. Claiming that it is protective against FAdV-4 gives the mistaken impression that it can protect against any strain of that serotype, and that was not evaluated.
•    The authors point out the limitation of not having compared fiber-2 individually. The same applies to hexon. Furthermore, the possibility that the combined formulation may be detrimental compared to the individual formulations cannot be ruled out.
•    Another very important limitation that must be considered is the lack of evaluation of the vaccine candidate against different strains of serotype 4 (of moderate and high virulence).
•    Conclusions: Here and in other sections of the manuscript, there is an overestimation of the study results. Terms such as "strong," "robust," and "greater protective efficacy" seem exaggerated based on the preliminary results shown (validation in small groups) and should be adjusted to reflect the actual findings.

Author Response

We thank the reviewer for the careful evaluation of our manuscript and for the constructive comments, which have helped us improve clarity, accuracy, and scientific balance. All comments have been carefully considered, and the manuscript has been revised accordingly, as detailed below.

Major Observation 1

The authors state that current commercial vaccines offer limited protection, but provide no evidence that their vaccine is comparable.

Authors’ response

We agree with the reviewer that direct comparison with commercially available vaccines was not performed and that this represents a limitation of the present study. The Introduction and Discussion have been revised to clarify that our statement regarding commercial vaccines is based on previously published studies and field reports, rather than on direct experimental comparison. We now explicitly state that no conclusions regarding comparative efficacy can be drawn from this study and that such comparisons should be addressed in future investigations.

Major Observation 2

The authors overestimate the application to FAdV-4, since only homologous challenge was performed.

Authors’ response

We fully agree and have revised the title, abstract, Discussion, and Conclusions to avoid overgeneralization. The manuscript now clearly states that the protective efficacy was evaluated only against the homologous FAdV-4 strain used for antigen production. All statements implying broader protection across FAdV-4 strains have been removed or rephrased to accurately reflect the experimental scope.

Other Comments

Pathological description of HHS

The authors should provide a pathological description of HHS and differentiate virulence forms.

Authors’ response

A concise pathological overview of HHS has been added to the Introduction, describing the typical lesions associated with classical and hypervirulent FAdV-4 infections. Where appropriate, distinctions between mild and severe disease presentations are now highlighted based on published literature.

Line 44

“Molecular genotyping” is more appropriate.

Authors’ response

Corrected as suggested.

Lines 75–78

Epidemiological support must include published studies.

Authors’ response

Additional epidemiological references have been incorporated to support the statements in this section. Unpublished surveillance data are now clearly identified as supplementary and not used as sole evidence.

Line 113

Add reference defining the virulence of the strain used.

Authors’ response

The appropriate reference defining the virulence characteristics of the challenge strain has been added.

Section 2.10.1 – Challenge conditions and animal husbandry

Authors’ response

This section has been expanded to include detailed information on housing conditions, biosecurity measures, separation of experimental groups, temperature, humidity, lighting, and management practices. These additions clarify that environmental and cross-contact confounders were minimized.

Section 2.10.2 – Organ selection

Why were intestinal segments not included?

Authors’ response

We appreciate the reviewer's thoughtful insights. We wish to clarify that the serotype targeted in this study—adenovirus type 4 (FAdV-4)—exhibits a highly organ-specific pathogenic spectrum. According to literature and consensus in this field (key references include Schachner et al., 2018; Yang Cheng-Hui et al., 2017), the characteristic fatal lesions of FAdV-4 infection are pericardial effusion and hepatitis, with the liver and heart as primary target organs. Although the virus enters via the oral route, the gut typically serves only as the initial portal of entry and does not develop significant lesions such as encephalitis (which may occur with certain virulent strains) or severe enteritis. Therefore, in this study, we focused on its recognized core target organs (liver, heart) and included brain tissue to investigate its potential neurotropism. This design aligns with the pathological focus of this specific pathogen.

Figures S1–S3

Presence of ~100 bp bands suggests possible byproducts or contamination.

Authors’ response

A constant band of approximately 100 bp was observed in all PCR reactions, including both positive and negative controls. Upon comparison with the no-template control, this band was identified as a primer dimer.

Despite the presence of primer dimers, their size differed significantly from the target fragment (690 bp). Furthermore, specific target fragments were obtained through gel extraction and purification, ensuring the reliability of subsequent experiments such as cloning and sequencing. However, due to the passage of time and the fact that this gel was not retained at the time, we are unable to provide you with the verification gel from that period.

Figure S8

Additional band observed in purified hexon-L1 protein.

Authors’ response

Thank you for pointing out the details in the protein purification figure. We agree that a smaller band is present below the hexameric L1 main band. This is a common byproduct in recombinant protein expression and purification, typically arising from partial degradation or translational truncation. Importantly, the identity (confirmed by mass spectrometry) and function of the main band have been validated, and its purity is sufficient to support all downstream functional experiments in this study. We have added this clarification to the main text. Thank you for your valuable feedback.

Table 5

Virus shedding not evaluated in controls at all dpi.

Authors’ response

Thank you very much for your question. We evaluated viral shedding in the control group during the actual experimental process, though this may not have been clearly reflected in the table. We have added a footnote to better explain the results.

Discussion – Repetition of results

Authors’ response

The Discussion has been thoroughly revised to eliminate repetition of Results. Redundant descriptive sentences have been removed and replaced with interpretation-focused discussion.

Lines 501–503

Supporting references are inappropriate (e.g., Salmonella).

Authors’ response

We thank the reviewer for identifying this issue. All inappropriate or unrelated references have been removed and replaced with relevant literature. Similar instances throughout the manuscript have been corrected.

Lines 504–505

Overgeneralized protection claims.

Authors’ response

These claims have been revised to clearly state that protection was demonstrated only against the homologous challenge strain. Overgeneralized language has been removed throughout the manuscript.

Limitations of antigen comparison

Lack of individual fiber-2 and hexon comparison; possible detrimental effects of combination.

Authors’ response

We agree and have expanded the Limitations subsection to explicitly state that the individual contributions of hexon-L1 and fiber-2 were not evaluated, and that potential antagonistic effects of the combined formulation cannot be excluded. This point is now clearly acknowledged.

Lack of heterologous strain evaluation

Authors’ response

This important limitation is now explicitly stated in the Discussion. The need to assess vaccine efficacy against multiple FAdV-4 strains of varying virulence is highlighted as a critical next step.

Conclusions – Overestimation of results

Authors’ response

The Conclusions have been carefully revised to adopt more conservative terminology. Words such as “strong,” “robust,” and “greater” have been replaced with language that accurately reflects the preliminary nature of the findings and the limited sample size.

Final statement

We sincerely thank the reviewer for the insightful comments, which have significantly improved the scientific rigor, clarity, and balance of the manuscript. The revised version now presents a more accurate and appropriately cautious interpretation of the findings.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Chu et al., “A Bivalent Subunit Vaccine Candidate Against Fowl Adenovirus Serotype 4, reports the development an evaluation of a bivalent vaccine against fowl adenovirus serotype 4 in chickens.

The hypothesis is that a bivalent vaccine formulation, combining recombinant hexon L1 and fiber 2 proteins, will provide superior protection compared to the monovalent vaccine based solely on the fiber 2 protein (which has previously been shown to confer strong protection against AdV4 challenge).

To validate or refute this hypothesis, the authors must conduct challenge experiments in chickens immunized with both the monovalent and bivalent vaccine candidates. However, in the present study only two dose levels of the bivalent formulation were evaluated; therefore, the hypothesis cannot be adequately tested.

In my opinion, such an experiment is essential for publication.

Author Response

Response to Reviewer n. 2

We thank the reviewer for this important and constructive comment. We fully agree that a direct head-to-head comparison between the bivalent (hexon-L1 + fiber-2) formulation and a fiber-2-only vaccine would be the most rigorous approach to conclusively demonstrate additive or synergistic protection.

The primary objective of the present study, however, was not to demonstrate superiority over fiber-2 alone, but rather to establish the feasibility, safety, and protective efficacy of a bivalent subunit formulation combining two major antigenic determinants of FAdV-4. Our working hypothesis was that a bivalent construct could elicit strong, dose-dependent protection and reduce viral dissemination and shedding, which we demonstrate clearly at the 20 μg dose.

We acknowledge that the absence of a fiber-2-only control group limits direct conclusions regarding the incremental contribution of hexon-L1. To address this limitation transparently, we have:

1. Revised the Introduction and Discussion to avoid claims of superiority over fiber-2 alone.
2. Explicitly stated this limitation in the “Limitations of the Study” section.
3. Added a Future Research Directions paragraph indicating that comparative studies including fiber-2-only vaccination are currently planned and will be essential to determine synergistic or additive effects.

Importantly, despite this limitation, the bivalent vaccine demonstrated complete protection, absence of virus shedding, and lack of detectable viral antigen in tissues at the higher dose, which are highly relevant endpoints for disease control and field application. We therefore believe the current dataset provides valuable and publishable evidence supporting further development of this bivalent vaccine concept.

The manuscript has been revised accordingly to reflect these clarifications and to align conclusions strictly with the experimental data presented.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a useful and well-written manuscript that can be published with minimal changes. The authors have presented their efforts to develop a vaccine against adenovirus infection of poultry, which a significant and potentially devastating infection of these species.

 

Some minor points are listed below in order to improve the final paper.

 

1. The justification for developing this vaccine is not strong, therefore please present more strong arguments.
2. Please explain the gap that will be filled by publishing this paper.
3. The Introduction is on the long side, hence please consider dividing into sub-sections – this may seem unusual, but not unheard of, and it will definitely improve the flow of reading in the final paper.
4. Please check the sequences in Table 1 to confirm lack of errors.
5. Please insert a new section in M&M to describe in great detail all the control material used in the study and all the control animals enrolled in the experiments and all the control procedures performed during this work.
6. The Results section lacks visualization really. You must include 4 or 5 more graphs to show the results in a summarized manner, which will help the readers to get a quick overview of the results.
7. Discussion, please divide into sections, at least 4-5 section to make easier the flow of reading.
8. Conclusions. Please close with a strong takeaway sentence.

 

 

Overall. Excellent manuscript for immediate acceptance after making the above changes.

Author Response

Point-by-point reply to Reviewer #3

Reviewer comment 1

The justification for developing this vaccine is not strong, therefore please present more strong arguments. Please explain the gap that will be filled by publishing this paper.

Authors’ response

We thank the reviewer for this valuable suggestion. The Introduction has been revised to more clearly articulate the scientific and practical rationale for developing this vaccine. Specifically, we now emphasize: (i) the limitations of currently available live-attenuated and inactivated FAdV-4 vaccines, including safety concerns and incomplete prevention of viral shedding; (ii) the lack of experimentally validated bivalent subunit vaccine strategies for FAdV-4; and (iii) the need for vaccines capable of reducing both disease and environmental viral load.

The gap addressed by this study is the experimental evaluation of a bivalent hexon-L1 and fiber-2 subunit vaccine, including its effects on pathology, tissue viral distribution, and virus shedding following virulent challenge. This has now been explicitly stated in the revised Introduction.

Reviewer comment 2

The Introduction is on the long side, hence please consider dividing into sub-sections.

Authors’ response

We agree with the reviewer and have restructured the Introduction into clearly defined sub-sections to improve readability and logical flow. The revised Introduction now includes sub-headings addressing: (i) epidemiology and impact of FAdV-4; (ii) current vaccine strategies and their limitations; and (iii) rationale for a bivalent subunit vaccine approach.

Reviewer comment 3

Please check the sequences in Table 1 to confirm lack of errors.

Authors’ response

The primer sequences reported in Table 1 have been carefully re-checked against the reference genome of strain SDLC202009 and verified for accuracy. No errors were identified. This confirmation is now noted in the revised manuscript.

Reviewer comment 4

Please insert a new section in M&M to describe in great detail all the control material used in the study and all the control animals enrolled in the experiments and all the control procedures performed during this work.

Authors’ response

We appreciate this suggestion and have added a dedicated subsection in the Materials and Methods entitled “Control Materials, Animals, and Procedures”. This section now details the negative control group, saline injections, pre-screening of SPF chickens, procedural controls during vaccination and challenge, and negative controls used in PCR, histopathology, and immunohistochemistry.

Reviewer comment 5

The Results section lacks visualization really. You must include 4 or 5 more graphs to show the results in a summarized manner.

Authors’ response

We agree that additional visualization improves clarity. The Results section has been enhanced with new summary figures.

Reviewer comment 6

Discussion, please divide into sections, at least 4-5 section to make easier the flow of reading.

Authors’ response

The discussion has been completely reorganised to make it easier to read.

Reviewer comment 7

Conclusions. Please close with a strong takeaway sentence.

Authors’ response

We have revised the Conclusions section and added a clear and strong final takeaway sentence highlighting the relevance of the findings for future FAdV-4 vaccine development and disease control in poultry.

 

Reviewer 4 Report

Comments and Suggestions for Authors

Review of microbiolres-4066988 A Bivalent Subunit Vaccine Candidate Against Fowl Adenovirus Serotype 4

 

My questions and queries

 

• Line 63 – put references
• Line 91 – “The most effective outcomes in chicken protection against HHS via subunit vaccines have been realized by FAdV-4 fiber2 vaccination” – put references
• Line 265 serological test and results with it – line 420 – I do not know how many day past between those analyses ?
• Line 305 – better description of the Figure S5 is needed – figures and tables must be readable without text.
• Line 307 – better description of the Figure S6 and S7 is needed – figures and tables must be readable without text.
• Figure 2 - the quality of the pictures is weak,
• Line 367 – I know that there was limited number of birds, but is better to give information how many birds show histopathological changes – necrosis, oedema, ….…… in all groups in all examined organs. – You can add it as supplements
• Line 368 and other lines- There is no myocardial fiber oedema – change into myocardial oedema,
• If you diagnosed degeneration you have to state what kind of degeneration have you diagnosed: vacuolar, fatty, parenchymatous…..
• Line 379 – the quality of the pictures is not acceptable. Higher magnification is needed. Show us the basophilic inclusion bodies in hepatocytes nucleus.
• Line 381 - better description of the Figure 3 is needed – figures and tables must be readable without text.
• Line 393 – I do not see any reaction. The quality of pictures is tragic.
• Line 393 – figure F – I do not believe it is pancreas
• Line 395 - better description of the Figure 4 is needed – figures and tables must be readable without text.
• Line 404 – “high viral concentrations were consistently detected” – how much ?
• Line 418 – table 5 – give info under table that in control group there is no data because birds were dead.
• Line 429 – figure 5 – days – look earlier
• Discussion chapter – long beginning – it is more like introduction than discussion; all your results have to be discussed – more discussion is needed.
•  

 

Major review is needed

Author Response

Reviewer #4 – Point-by-Point Response

Comment 1 (Line 63): Put references

Reviewer’s comment:

Line 63 – put references.

Response:

We thank the reviewer for this observation. Appropriate references supporting the statement at line 63 have now been added.

Comment 2 (Line 91): Fiber-2 vaccination statement – put references

Reviewer’s comment:

Line 91 – “The most effective outcomes in chicken protection against HHS via subunit vaccines have been realized by FAdV-4 fiber2 vaccination” – put references.

Response:

We agree with the reviewer. The statement has now been supported by multiple published studies demonstrating the protective efficacy of FAdV-4 fiber-2-based subunit vaccines.

Comment 3 (Lines 265–420): Serology timing unclear

Reviewer’s comment:

Line 265 serological test and results with it – line 420 – I do not know how many days passed between those analyses?

Response:

We appreciate this important comment. The timing of serum sampling relative to immunization and challenge was not sufficiently clear and has now been explicitly stated.

Comment 4 (Line 305): Figure S5 description insufficient

Reviewer’s comment:

Better description of Figure S5 is needed – figures must be readable without text.

Response:

We agree and have expanded the figure legend to provide a standalone description of the experimental conditions and results.

Comment 5 (Line 307): Figures S6 and S7 description insufficient

Reviewer’s comment:

Better description of Figures S6 and S7 is needed.

Response:

We thank the reviewer. The legends of Figures S6 and S7 have been revised to allow full interpretation without referring to the main text.

Comment 6 (Figure 2): Poor image quality

Reviewer’s comment:

Figure 2 – the quality of the pictures is weak.

Response:

We acknowledge this limitation. Figure 2 has been replaced with higher-resolution images with improved contrast and sharpness.

Comment 7 (Line 367): Number of birds with histopathological lesions

Reviewer’s comment:

Better to give information how many birds show histopathological changes – add as supplements.

Response:

We agree. Quantitative data on the frequency of histopathological lesions have now been added as supplementary material.

Comment 8 (Line 368): Terminology – myocardial fiber oedema

Reviewer’s comment:

There is no myocardial fiber oedema – change into myocardial oedema.

Response:

We thank the reviewer for this correction. The terminology has been revised throughout the manuscript.

Comment 9: Type of degeneration must be specified

Reviewer’s comment:

If you diagnosed degeneration you have to state what kind.

Response:

We fully agree and have now specified the type of degeneration observed.

Comment 10 (Line 379): Histology image quality and inclusion bodies

Reviewer’s comment:

Higher magnification is needed. Show basophilic inclusion bodies in hepatocyte nuclei.

Response:

We acknowledge this concern. High-magnification images highlighting intranuclear basophilic inclusion bodies have now been added

Comment 11 (Line 381): Figure 3 description insufficient

Reviewer’s comment:

Better description of Figure 3 is needed.

Response:

The figure legend has been expanded to fully describe lesions, organs, magnification, and group allocation.

Manuscript revision:

• Add:
• Magnification
• Type of lesions per panel
• Staining method (HE)

Comment 12 (Line 393): No visible reaction, poor IHC quality

Reviewer’s comment:

I do not see any reaction. The quality of pictures is tragic.

Response:

We acknowledge the reviewer’s concern and have replaced the immunohistochemical images with higher-contrast, higher-resolution images.

Comment 13 (Line 393, Figure F): Pancreas identification doubtful

Reviewer’s comment:

Figure F – I do not believe it is pancreas.

Response:

We thank the reviewer for this important observation. The tissue identification has been re-checked.

Comment 14 (Line 395): Figure 4 description insufficient

Reviewer’s comment:

Better description of Figure 4 is needed.

Response:

The legend has been revised to ensure Figure 4 is interpretable independently of the text.

Manuscript revision:

• Specify:
• Antibody used
• Positive staining appearance
• Magnification
• Group assignment per panel

Comment 15 (Line 404): “High viral concentrations” – how much?

Reviewer’s comment:

“High viral concentrations were consistently detected” – how much?

Response:

We agree that this wording was imprecise. The statement has been revised to reflect PCR-based detection rather than quantitative viral load.

Comment 16 (Line 418, Table 5): Missing control data explanation

Reviewer’s comment:

Give info under table that in control group there is no data because birds were dead.

Response:

We thank the reviewer. This clarification has been added to the table footnote.

Comment 17 (Line 429, Figure 5): Sampling days – look earlier

Reviewer’s comment:

Figure 5 – days – look earlier.

Response:

We agree and have corrected the timeline representation.

Comment 18 (Discussion): Too introductory, results not sufficiently discussed

Reviewer’s comment:

Discussion chapter – long beginning – more discussion is needed.

Response:

We appreciate this critical comment. The Discussion has been substantially revised to reduce introductory content and to focus on interpretation of the present results.

Final statement

Response:

We thank Reviewer #4 for the thorough and constructive critique. All comments were carefully addressed through textual revision, figure replacement, improved terminology, and addition of supplementary quantitative data. These changes have significantly improved the clarity, rigor, and scientific value of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have responded to or justified the comments made in the previous round of review appropriately.

Author Response

We thank the reviewer for enhancing the value of our manuscript and for welcoming our additions and corrections made in the previous round.

Reviewer 2 Report

Comments and Suggestions for Authors

I have carefully reviewed the authors’ response and the revised version of the manuscript. The principal limitation remains the absence of a control group immunized with the fiber 2 protein alone. I must reiterate my previous assessment that this constitutes a major experimental flaw. Without such a comparison to demonstrate any advantage over the single-protein candidate, the rationale for proposing a bivalent vaccine is not proved. I therefore strongly recommend that the authors conduct this comparative experiment prior to publication.

Author Response

We thank the reviewer for his contribution.

Nothing to add to what was said in the previous round.

Reviewer 3 Report

Comments and Suggestions for Authors

All the issues raised were duly addressed. No further comments.

Author Response

We thank the reviewer for enhancing the value of our manuscript and for welcoming our additions and corrections made in the previous round.

Reviewer 4 Report

Comments and Suggestions for Authors

Minor corrections 

Table S4 - delete it. Authors already mentioned about lesions in table S3 and number of lesions in organs is not the same (ex pancreas 4/10 in table S3 and S4)

 

Figure 4 - delete or explain arrows 

Author Response

We thank the reviewer for enhancing the value of our manuscript.
Table S4 has been deleted.
We have added a description of the arrows in Figure 4.