Immunomodulatory Effects of Escherichia coli Phage GADS24 on Human Dendritic Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Please clarify the exact residual LPS levels in each treatment group, especially for the phage-only and phage + lysate conditions.

 

 

2. Add precise p-values or significance markers in all figure legends and clarify which comparisons were tested.

 

 

3. Did you test heat-inactivated phage as a control? This would help confirm if the effects are due to the phage itself.

 

 

4. Please include a gating strategy figure or describe it more clearly in the methods.

 

 

5. Briefly discuss how these in vitro findings could inform in vivo or clinical applications of phage therapy.

 

 

6. Improve labeling and color contrast in flow cytometry figures to make data easier to interpret.

Author Response

Response to Reviewer 1 Comments:

We would like to thank the esteemed reviewer 1 for the thoughtful and constructive feedback provided on our manuscript. We greatly appreciate the detailed comments and suggestions, which have significantly improved our manuscript. Below, we provide point-by-point responses, and modifications in the manuscript are highlighted in yellow colour

Comment 1: Clarify residual LPS levels.

Response: Thank you for this valuable comment. We have clarified this in Section 2.6.2 of the Methods. Subsection" DC generation and treatment ", page 5.

• Phage-only: "Residual LPS was measured at 0.04 EU/mL.", line 193.
• Phage + lysate: "Residual LPS was measured at 0.37 EU/mL.", lines 194-195.
• It has also been mentioned in the discussion section page 16, lines 513-516)

Comment 2: Add precise p-values or significance markers in all figure legends and clarify which comparisons were tested

Response: Thank you for this valuable suggestion. To clearly illustrate statistical significance, we emphasize the following:

• Figures 1 and 2 primarily depict descriptive results; thus, statistical comparisons were not applicable (Figure 1). Plaque assay results demonstrating lysis efficiency at different phage dilutions. This figure is descriptive; no statistical comparisons were applicable (Figure 2). Morphological differences of dendritic cells upon treatments were visualized using inverted microscopy. Images are representative of three independent donors. No statistical comparisons were applicable for morphological observations.
• Figures 3–7: We would like to clarify that the dot plots and histograms presented in Figures 3–7 serve as representative flow cytometry readings for visualizing the expression profiles of specific surface markers. These figures are illustrative, not statistical. The complete statistical analysis, including p-values and group comparisons, is summarized in Figure 8, which consolidates all quantitative data derived from multiple experimental replicates. As stated in the legend of Figure 8, all statistical comparisons were performed against the untreated control group. We have now clarified this point explicitly in the figure legends to prevent misinterpretation, as requested.
• Figures 9-10 already contain significant markers.

Comment 3: Did you test heat-inactivated phage as a control? This would help confirm if the effects are due to the phage itself.

Response: We thank the reviewer for this important suggestion. In our experimental design, heat-inactivated phage was not tested as an individual control. However, the phage used in the bacterial lysate preparation was thoroughly inactivated by a mild heat treatment at 70 °C for 30 minutes, as described in the methods (Section 2.3, pages 3-4, and lines 141-145). This inactivation step was specifically introduced to eliminate residual active phage from the lysate fraction and was verified via spot assay to ensure complete loss of lytic activity. To assess the immune-modulatory role of the phage itself, fresh purified (active) phage was added back to the inactivated lysate during co-treatment experiments. This allowed us to distinguish the immunological effects of phage alone, lysate alone, and their combination. While a dedicated “heat-inactivated phage only” control could provide further granularity, we believe our current design and phage inactivation verification address the primary concern regarding phage-specific activity. We have clarified this point in the Methods section.

Comment 4: Please include a gating strategy figure or describe it more clearly in the methods.

Response: Thank you for this valuable recommendation. To address your comment, we have clarified our gating strategy more explicitly in the Methods section of the manuscript (subsection 2.7, page 5, lines 203-208), the Results section (subsection 3.4, pages 8-9, lines 299 and 303, and lines 309-317).  Additionally, we have explicitly revised Supplementary Figure S1 by clearly labeling panels as (A) and (B) directly within the figure. Panel A represents the unstained control used to establish baseline fluorescence, while Panel B demonstrates representative gating of positively stained dendritic cells (HLA-DR). The corresponding figure legend clearly describes each labeled panel and clarifies the gating strategy applied consistently throughout our analysis (page 20, lines 714-719).

Comment 5: Briefly discuss how these in vitro findings could inform in vivo or clinical applications of phage therapy.

Response: We sincerely thank the reviewer for highlighting this essential point. To address this clearly, we have expanded the Discussion section of our manuscript to explicitly discuss the potential implications of our in vitro results for future in vivo and clinical studies. Specifically, we have added a clear statement outlining how the observed immunomodulatory effects of bacteriophage GADS24 on dendritic cells may inform and guide subsequent preclinical and clinical phage therapy studies against multidrug-resistant bacterial infections (pages 18-19, lines 656-667).

Comment 6: Improve labeling and color contrast in flow cytometry figures to make data easier to interpret.

Response: Thank you very much for this valuable suggestion. We fully acknowledge the importance of clear and visually distinguishable flow cytometry data presentation. In response, we have carefully revised the labeling and improved the color contrast in all flow cytometry figures (Figures 3–7).

We sincerely thank Reviewer 1 for their thorough and constructive feedback. Your comments have greatly improved the clarity, scientific rigor, and overall quality of our manuscript. We have carefully addressed each point raised, and we hope that the revised version now meets the journal's expectations. We respectfully submit it for your reconsideration.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In this study author evaluated the effects of GADS24 on the phenotypic maturation and cytokine secretion profiles of human monocyte-derived dendritic cells by comparing GADS24 to bacterial lysates. In the following section author will find few comments for their consideration. 

Comments:

1. Page 3, line 94: Briefly mention how the spot assay was done and provide the reference.
2. Page 3, section 2.3: This method does not make sense. please rewrite it using a method paper and reference the paper.
3. Page 3, line 102-104: Author can have a heading "strains and phage" and mention from where they were obtained to avoid writing again and again.
4. Page 3 line107: This has been mentioned in the earlier paragraph.
5. Page 3 line111-112: please explain how the active phage particles were removed by 0.22uM filter. This filter can only remove bacteria not the phage as phage size is in nanometer range.
6. Page 3 line 112-113: why do you have to add purified phage in the bacterial lysate. please explain?
7. Page 3 line 114: Reference 14 is a case report. please use a method paper for the method.
8. Page 3 line 115: please explain the LPS removal method briefly.
9. Page 3 line 119-120: this sentence looks incomplete.
10. Page 3 line 123-124: This sentence has already been mentioned at the start of the paragraph.
11. Page 3 section 2.5: phage infection range has already mentioned in heading 2.2.
12. Page 5 line 203-204: this sentence has already been mentioned in the method section.
13. Page 5 table 1 needs formatting.
14. Page 5 Section 3.2: please mention the minimum effective titer of the phage.
15. Page 6 line 255: Author mentioned “phage concentrations were 10-9 and 10-10..” is this the concentration or dilution factor of phage stock?
16. Page 7 line 267-269: these sentences has been covered in the figure legend.

 

Author Response

Response to Reviewer 2 Comments:

We would like to express our sincere gratitude to Reviewer [2] for their valuable time, thoughtful feedback, and constructive suggestions. Your insightful comments have significantly improved the clarity, methodology, and scientific impact of our manuscript. Below, we provide a detailed response to each of your points and describe the corresponding changes made to the manuscript. The modifications are highlighted in gray colour.

Comment 1: Page 3, line 94: Briefly mention how the spot assay was done and provide the reference.

Response: We thank the reviewer for this helpful comment. The description of the spot assay procedure was already included in the Materials and Methods section (subsection 2.2, Page 3, lines 129-134), and a supporting reference (Bonilla et al. [17]) was provided. For clarity, we have now ensured that the method is concise and properly formatted, and the reference citation has been retained in the revised.

Comment 2: Page 3, section 2.3: This method does not make sense. Please rewrite it using a method paper and reference the paper.

Response: We thank the reviewer for this critical comment. In response, we have rewritten Subsection 2.3 (pages 3- 4, lines 135-149) to clarify the bacterial lysate preparation protocol based on an established method and cited the appropriate reference (Bonilla et al., 2016 reference [17]). The strain E. coli NRT114 was kindly provided by Ms. Sana Khalifa Alsheikh at the King Fahd Medical Research Center (KFMRC), King Abdulaziz University, and the reference of this strain is [17].

Comment 3: Page 3, line 102-104: The Author can have a heading "strains and phage" and mention from where they were obtained to avoid writing again and again.

Response: We thank the reviewer for this helpful suggestion. In response, we have added a new subsection titled “2.1. Bacterial Strains and Phage” to consolidate the source and description of E. coli strain NRT114, the novel lytic phage GADS24, and the panel of antibiotic-resistant E. coli strains used in host range testing. This reduces redundancy and improves the clarity of the Materials and Methods section on page 3.

Comment 4: Page 3, line 107: This has been mentioned in the earlier paragraph.

Response: We thank the reviewer for this observation. While we understand the concern regarding potential repetition, we respectfully clarify that the methods described refer to two distinct experimental procedures. The earlier paragraph describes the plaque assay used for phage titration, while the subsequent section details the spot assay method used for host range evaluation. This was stated on page 3, lines 111 and 130.

Comment 5: Page 3 line111-112: please explain how the active phage particles were removed by the 0.22 μm filter. This filter can only remove bacteria, not the phage, as phage size is in the nanometer range.

Response: We thank the reviewer for this necessary clarification. While 0.22 µm filtration removes bacterial contaminants, we agree that phage particles, due to their nanometer size, can pass through. Therefore, we have now clarified a heat inactivation step at 70°C for 30 minutes after filtration, which is commonly used to inactivate tailed phages while preserving the immunogenicity of the bacterial lysate. We have also confirmed the absence of infectious phages via spot assay. This modification has been incorporated into Section 2.3 of the revised manuscript ( pages 3-4, lines 135-151).

 

Comment 6: Page 3 line 112-113: why do you have to add purified phage in the bacterial lysate. please explain?

Response: We thank the reviewer for this insightful question. The addition of purified phage to the sterile bacterial lysate was performed to specifically assess the combined effect of bacteriophage components and bacterial debris on dendritic cell maturation and cytokine responses. This allowed us to distinguish between the immunomodulatory effects of phage alone, bacterial lysate alone, and the synergistic interaction of a calculated dose of the two. Such co-treatment models are increasingly used to mimic complex microbe-host interactions and have been described in phage immunology studies. We have clarified this rationale in the revised manuscript (Subsection 2.3, page 4, lines 149-151).

 

Comment 7: Page 3 line 114: Reference 14 is a case report. please use a method paper for the method.

Response: We thank the reviewer for this critical correction. Reference 14, previously a case report, has been replaced with a recent methodological paper by Jacobs-Sera et al. (2012), which outlines standardized protocols for phage propagation, purification, and quantification. This update ensures methodological accuracy and proper citation without altering the numbering of the reference list.

 

Comment 8: Page 3 line 115: please explain the LPS removal method briefly.

Response: We thank the reviewer for pointing this out. In response, we have now added a brief explanation of the LPS removal procedure used in our phage preparation. The method involved 1-octanol extraction followed by dialysis, which are commonly used steps to reduce endotoxin contamination in phage samples. The revised description has been added to Page 4, lines 156-160 and 162-166 of the Methods section ( subsection 2.4).

Comment 9: Page 3, lines 119-120: This sentence looks incomplete.

Response: We thank the reviewer for pointing out this grammatical issue. The sentence on lines 119–120 has now been rephrased for clarity and completeness. It now correctly states that 1-octanol was used for LPS removal from the phage preparation, referencing the appropriate methodology. This revision appears in Section 2.4 of the updated manuscript (page 4, lines 153-155).

 

Comment 10: Page 3 line 123-124: This sentence has already been mentioned at the start of the paragraph.

Response: We thank the reviewer for pointing out this redundancy. The sentence on lines 123–124 has already been introduced earlier in the paragraph and has now been removed according to the previously mentioned modifications.

 

 

Comment 11: Page 3 section 2.5: phage infection range has already mentioned in heading 2.2.

Response: We thank the reviewer for the observation. However, we would like to clarify that the spot assay is the experimental method used across multiple sections of the study. At the same time, host range refers to the biological outcome derived using this method. To improve clarity and avoid redundancy, we have maintained and switched separate sections: one dedicated to the spot assay protocol and another to describe its application for host range determination. This separation reflects the methodological vs. interpretative focus of each part.

 

Comment 12:  Page 5 line 203-204: this sentence has already been mentioned in the method section.

Response: We thank the reviewer for noting this redundancy. The sentence on lines 203–204 repeated information already detailed in the Methods section and has now been removed to avoid unnecessary duplication and improve clarity.

Comment 13:  Page 5 table 1 needs formatting.

Response: We thank the reviewer for noting this. The table formatting is adjusted (page 6, table 1)

 

Comment 14:  Page 5 Section 3.2: please mention the minimum effective titer of the phage.

Response: We thank the reviewer for this insightful comment. The minimum effective titer of the GADS24 phage used in our experiments was 1 × 10⁸ PFU/mL, as specified in the Materials and Methods section (Section 2.3).

Comment 15:  Page 6 line 255: Author mentioned “phage concentrations were 10^-9 and 10^-10..” is this the concentration or dilution factor of phage stock?

Response: We thank the reviewer for pointing out this ambiguity. The values “10⁻⁹ and 10⁻¹⁰” refer to the dilution factors of the original phage stock used in the plaque assay, not absolute concentrations. We have revised the sentence in the Results section (Subsection 3.2, Pages 6-7, lines 260-261, and 266, 268, and 269) to indicate this.

 

Comment 16:  Page 7 line 267-269: these sentences has been covered in the figure legend.

Response: We thank the reviewer for this helpful observation. The sentences on lines 267–269 repeated information already stated in the figure legend. These have now been removed and replaced with a concise, interpretive reference to the figure (page 7, lines 284-286).

 

Finally, we sincerely thank the reviewer [2] for their thoughtful and constructive comments. Your detailed feedback has helped us improve the clarity, methodological accuracy, and scientific presentation of our manuscript. We have addressed all comments carefully and revised the manuscript accordingly. We greatly appreciate your contribution to enhancing the quality of our work.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The current study by Aldahlawi et al., titled "Immunomodulatory Effects of Escherichia coli Phage GADS24 on Human Dendritic Cells" investigates the impact of GADS24, a novel lytic bacteriophage targeting E. coli, on human monocyte-derived dendritic cells (DCs). The results demonstrate that treatment with bacterial lysate significantly upregulated DC maturation markers, whereas GADS24 phage alone induced a moderate increase in these markers, reduced IL-10 secretion, and elevated IL-12p70 production. Notably, the combination of bacterial lysate and phage attenuated the maturation response compared to lysate treatment alone. These findings suggest that GADS24 possesses both antibacterial properties and immunomodulatory capabilities, influencing DC maturation and cytokine secretion. Given its dual antimicrobial and immunoregulatory functions, GADS24 holds potential as an adjunctive therapeutic agent against multidrug-resistant (MDR) bacterial infections. However, further in vivo studies are necessary to validate these in vitro observations. Despite the promising results, the manuscript requires substantial revisions, as several unresolved questions remain regarding the experimental design and methodology, necessitating clarification and refinement to strengthen the study's conclusions.

1. Add background on other bacteriophages targeting ESKAPEE bacteria to contextualize your study.
2. Strengthen the justification for studying a novel bacteriophage—why is this specific phage significant compared to existing research?
3. The study needs a stronger argument for its uniqueness. Thousands of papers have isolated and studied lytic bacteriophages how does your work stand out?
4. Was this phage newly isolated, or were only its lytic effects examined?
5. Provide evidence that this phage hasn’t been studied before (e.g., genomic comparison, database searches).
6. Table 1. How were the strains differentiated? Were any duplicates present? Include identification methods (e.g., biochemical tests, sequencing) to ensure no repetition.
7. Line 210 (Phage Titration) will be more accurate.
8. Table 1. Were experiments replicated? If so, state the number of replicates for statistical validity.
9. Figure 1: remove handwritten notes and use digital labels.
10. Discussion part, Add comparative analysis (e.g., a table comparing your phage’s properties with known phages). Emphasize why this study matters how does it advance the field?
11. Supplementary Material: Remove if it doesn’t add value. Focus on essential data in the main text.
12. Ensure all figures/tables are polished and professionally formatted.

Author Response

Response to Reviewer 3 Comments:

We sincerely thank Reviewer 3 for their insightful and constructive comments, which have helped us identify areas requiring clarification and improvement. We have carefully addressed each point raised and made the corresponding revisions in the manuscript. Below, we provide a detailed, point-by-point response to all suggestions. Modifications in the manuscript are highlighted in blue.

Comment 1: Background on bacteriophages targeting ESKAPEE bacteria.

Response: Thank you for this insightful suggestion. We agree that providing background on phages targeting ESKAPEE pathogens strengthens the context of our study. In response, we have added new content to the Introduction section of the manuscript, highlighting key findings and recent advances in phage therapy research targeting ESKAPEE organisms, with a specific emphasis on E. coli and related multidrug-resistant strains (page 2, lines 64-79). This update incorporates new references 10-13.

Comment 2: Strengthen the justification for studying a novel bacteriophage—why is this specific phage significant compared to existing research?

Response: We thank the reviewer for this important observation. In response, we have expanded the Introduction and Discussion sections of the manuscript to articulate the novelty and relevance of the GADS24 bacteriophage clearly. Specifically, we now highlight that GADS24 is a newly isolated, lytic Escherichia coli phage with no previously published immunological profiling. Unlike most studies that focus solely on bactericidal properties, our study uniquely investigates GADS24’s immunomodulatory effects on human dendritic cells. This dual-functional characterization offers novel insights into the field of phage therapy, where understanding immune interactions is crucial for translational application (page 2, lines 80-89).

Comment 3: The study needs a stronger argument for its uniqueness. Thousands of papers have isolated and studied lytic bacteriophages how does your work stand out?

Response: We thank the reviewer for this important observation. While it is true that many studies have focused on isolating and characterizing lytic bacteriophages, our work is distinct in two key ways. First, GADS24 is a newly isolated lytic Escherichia coli phage whose genome has been deposited in GenBank (accession number OQ703618). To our knowledge, no prior reports have assessed its biological properties. Second, and more importantly, our study goes beyond classical bacteriophage characterization by evaluating the immunomodulatory effects of the phage on human monocyte-derived dendritic cells (DCs). This dual approach bridges bacteriophage biology and immunology, contributing novel insight into how phage therapy may impact host immune responses. We have now emphasized this uniqueness more clearly in both the Introduction (page 3, lines 98-101) and Discussion (page 15, lines 478-483) sections.

Comment 4: Was this phage newly isolated, or were only its lytic effects examined?

Response: We appreciate the reviewer’s question. GADS24 is a newly isolated lytic Escherichia coli phage that was purified from environmental samples collected at a poultry facility. While its lytic activity was confirmed, this study represents the first investigation of its immunomodulatory effects on human dendritic cells. To emphasize its novelty, we have revised the Introduction and Methods sections to clearly state that this phage was newly isolated as part of this work, and its genome has been deposited in GenBank under accession number OQ703618 (Page 3, lines 95-96, and page 3, lines 104-106).

Comment 5: Provide evidence that this phage hasn’t been studied before (e.g., genomic comparison, database searches).

Response: We thank the reviewer for this important observation. GADS24 is a newly isolated lytic Escherichia coli bacteriophage. To confirm its novelty, we performed a comprehensive BLASTn search using its complete genome sequence against the NCBI nucleotide database. The search revealed no existing records with greater than 95% identity across the whole genome, supporting the conclusion that this phage had not been previously described or studied. Furthermore, the genome sequence of GADS24 was submitted by our research group and has been assigned the GenBank accession number OQ703618. To the best of our knowledge, this is the first report investigating the immunomodulatory effects of GADS24 or any data related to its genome or function. This clarification is provided in the discussion section, page 15, lines 478-483.

Comment 6: Table 1. How were the strains differentiated? Were any duplicates present? Include identification methods (e.g., biochemical tests, sequencing) to ensure no repetition.

Response: We thank the reviewer for this critical comment. All Escherichia coli strains listed in Table 1 were previously isolated and identified by the Special Infectious Disease Unit at the King Fahd Medical Research Center, King Abdulaziz University. The strains were confirmed to be unique using a combination of standard biochemical profiling and 16S rRNA gene sequencing. We have now clarified this methodology explicitly in the revised manuscript under the Materials and Methods section, and we have updated the Table 1 legend accordingly to reflect the identification procedures and ensure there was no duplication (page 3, lines 123-126, and page 6, lines 245-249). To clarify, host range testing (Table 1) was conducted using spot tests on a panel of antibiotic-resistant E. coli strains. Each strain was tested in triplicate to confirm reproducibility and prevent false positives or negatives. In contrast, phage titration was conducted only on the primary propagation host strain (E. coli NRT114).

Comment 7: Line 210 (Phage Titration) will be more accurate

Response: Thank you. The modification is done on page 6, line 257.

 

Comment 8: Table 1. Were experiments replicated? If so, state the number of replicates for statistical validity.

Response: We thank the reviewer for raising this critical point. We have now clarified this in the Materials and Methods section (page 4, lines 170-171) and updated the Table 1 legend to indicate the number of replicates performed (pages 5-6, lines 246-238, and lines 255-247).

Comment 9: Figure 1: remove handwritten notes and use digital labels.

Response: We sincerely thank the reviewer for this helpful suggestion aimed at improving the clarity and professionalism of Figure 1. In response, we have removed all handwritten annotations and replaced them with standardized, high-contrast digital labels for each panel (A–J). Each Petri dish is now clearly labeled with its corresponding dilution factor (10⁻¹ to 10⁻¹⁰) using consistent font size and formatting. The revised figure enhances the visibility of the plaques while ensuring clear identification of each sample (page 7, Figure 1).

Comment 10: Discussion part, Add comparative analysis (e.g., a table comparing your phage’s properties with known phages). Emphasize why this study matters, how does it advance the field?

Response: We thank the reviewer for the suggestion to include a comparative analysis in the Discussion. In response, we have incorporated a new paragraph comparing the biological and immunological features of GADS24 to several well-known E. coli phages (e.g., T4, phiEco32, vB_EcoM-VR20). This comparison is now presented in narrative form, highlighting how our study advances the field by integrating host immune response profiling, which is rarely addressed in traditional phage characterization (page 18, lines 620-637).

Comment 11: Supplementary Material: Remove if it doesn’t add value. Focus on essential data in the main text.

Response: We thank the reviewer for raising this point. We respectfully propose to retain Supplementary Figure S1, as it provides essential technical validation of the flow cytometry data presented in the main text. The figure illustrates the comprehensive gating strategy employed to define viable dendritic cells and quantify the expression of key surface markers (CD80, CD83, CD86, and HLA-DR). Inclusion of this figure ensures methodological transparency and reproducibility, which are crucial for immunological studies that rely on flow cytometric analysis. To clarify its relevance, we have now explicitly referenced Supplementary Figure S1 in the Methods section of the main manuscript ( pages 9, lines 311-319) and expanded the figure legend accordingly ( page 20, lines 7153-720).

 

Comment 12: Ensure all figures/tables are polished and professionally formatted.

Response: We thank the reviewer for the helpful reminder regarding visual quality and formatting. In response, we have carefully reviewed and revised all figures and tables to ensure consistency, clarity, and professional presentation. Specifically, we:

• Replaced all handwritten annotations in Figure 1 with clean digital labels.
• Standardized font size, line weights, and resolution across all flow cytometry plots (Figures 3–7).
• Ensured all image files meet high-resolution standards (≥300 dpi) suitable for publication.

 

 

We sincerely thank Reviewer 3 for their thorough and constructive feedback. Your comments have greatly improved the clarity, scientific rigor, and overall quality of our manuscript. We have carefully addressed each point raised, including expanding the literature review, clarifying the research hypothesis, providing genomic and endotoxin data, and refining figure presentation. We hope that the revised version now meets the journal's expectations and respectfully submit it for your reconsideration.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Aldahlawi and colleagues presents a study on how a T4-like coliphage affects human dendritic cells, specifically its immunomodulatory effects. This is important because infections from drug-resistant E. coli are becoming more common. The research shows the phage can change the immune system, suggesting it could be used with other antibiotics. Virologists and medical researchers should find the results interesting. The manuscript is well-organized, easy to follow, and the figures help explain the findings. Overall, it’s a good study that could be published in Biomedicines, but some revisions are needed before it’s ready.

Some notes:

1. The introduction sets the stage but doesn’t clearly state what the researchers expected to find (research hypothesis).
2. The literature review is weak, with a limited number of cited sources. It should be expanded to include more current reviews and articles (2020–2025) about how phages interact with the immune system.
3. Details about the cells used (sex, age, inclusion/exclusion criteria) from the three donors are missing. This information is needed to assess if the results can be repeated. The methods mention PI staining for flow cytometry, but the results don’t include data on cell viability after phage/lysate treatment, which is important for understanding the phage’s specific effects.
4. There’s no analysis or discussion of the phage’s genome or general properties. A short analysis should be included.
5. The authors mention a combined effect, but don’t explain how it works. The manuscript should discuss possible mechanisms. 
6. Although LPS reduction is mentioned, the exact values after purification (EU/mL) aren’t provided. This is important to prove the phage, not leftover LPS, is causing the observed effects.

Author Response

Response to Reviewer 4 Comments:

Thank reviewer 4 for their insightful and constructive comments, which have helped us identify areas requiring clarification and improvement. We have carefully addressed each point raised and made the corresponding revisions in the manuscript. Below, we provide a detailed, point-by-point response to all suggestions. Modifications in the manuscript are highlighted in green colour.

Comment 1: The introduction sets the stage but doesn’t clearly state what the researchers expected to find (research hypothesis).

Response: We thank the reviewer for this insightful suggestion. While the Introduction outlined the novelty and rationale of the study, we agree that the research hypothesis was not clearly stated. We have now added a concise statement at the end of the Introduction to present the hypothesis and expectations guiding this study clearly (page 2, lines 89-94).

Comment 2: The literature review is weak, with a limited number of cited sources. It should be expanded to include more current reviews and articles (2020–2025) about how phages interact with the immune system.

Response: We thank the reviewer for pointing out the need to strengthen the literature review. In response, we have expanded the Introduction to include recent publications (2020–2025) that address how bacteriophages interact with the immune system, particularly with innate immune cells such as dendritic cells. These references help contextualize our investigation of GADS24 within the emerging field of phage immunobiology (References 5, 10, 11, 12, and 13).

Comment 3: Details about the cells used (sex, age, inclusion/exclusion criteria) from the three donors are missing. This information is necessary to determine if the results can be replicated. The methods mention PI staining for flow cytometry, but the results don’t include data on cell viability after phage/lysate treatment, which is important for understanding the phage’s specific effects.

Response: We thank the reviewer for this valuable comment. We have now added detailed information about the human donors used to generate dendritic cells, including their age, sex, and the inclusion and exclusion criteria that were applied. These details are already provided in the Materials and Methods section, Subsection 2.6.1. Ethical approval to enhance reproducibility (page 4, lines 173-179).

Additionally, although PI staining was used to exclude dead cells during flow cytometry acquisition, we agree that reporting viability data is essential. We have now included a summary of PI-based viability percentages across all treatment groups in the Results section. These data confirm that phage and lysate treatments did not cause cytotoxic effects, supporting the specificity of the observed immunomodulatory responses (Page 5, lines 206-214).

Comment 4: There’s no analysis or discussion of the phage’s genome or general properties. A short analysis should be included.

Response: We thank the reviewer for this important suggestion. Although a complete genomic and phylogenetic analysis of GADS24 is part of a separate ongoing manuscript, we agree that providing a brief summary of the phage’s genomic features and general properties is necessary for context. Therefore, we have added a short genomic overview in the Discussion sections of the current manuscript (page 15, lines 485-498). This includes genome size, GC content, the number of predicted open reading frames (ORFs), and key gene functions relevant to lytic activity and structural components. The genome has been deposited in GenBank under accession number OQ703618.

Comment 5: The authors mention a combined effect, but don’t explain how it works. The manuscript should discuss possible mechanisms.

Response: We thank the reviewer for pointing this out. We agree that the concept of a combined effect between phage and lysate exposure on dendritic cells requires further explanation. In response, we have expanded the Discussion section to propose possible mechanisms based on current literature. We now discuss how phage structural proteins, residual bacterial debris, and associated pathogen-associated molecular patterns (PAMPs) might synergistically contribute to the observed immunomodulatory responses (page 18, lines 638-650).

Comment 6: Although LPS reduction is mentioned, the exact values after purification (EU/mL) aren’t provided. This is crucial to demonstrate that the phage, not leftover LPS, is responsible for the observed effects.

Response: We thank the reviewer for emphasizing the importance of LPS quantification. In response, we confirm that endotoxin levels in the final purified phage preparations were measured using the Limulus Amebocyte Lysate (LAL) chromogenic assay. The final endotoxin concentration was <0.05 EU/mL, a value well below the threshold known to activate human dendritic cells in vitro. This confirms that the observed effects in our study were due to the phage GADS24 and not residual LPS contamination. We have added this information to both the Materials and Methods (page 5, lines 193, 194, and 195) and Discussion sections of the revised manuscript (page 16, lines 513-517).

We sincerely thank Reviewer 4 for their thorough and constructive feedback. Your comments have greatly improved the clarity, scientific rigor, and overall quality of our manuscript. We have carefully addressed each point raised, including expanding the literature review, clarifying the research hypothesis, providing genomic and endotoxin data, and refining figure presentation. We hope that the revised version now meets the journal's expectations and respectfully submit it for your reconsideration.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

No further comments.