PONE-D-24-45377Boosting Immune Response Against Cervical Cancer: A Combined Approach Using Oncolytic Virus and Targeted TherapiesPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript investigates a novel therapeutic regimen for human papillomavirus (HPV)-associated cervical cancer, utilizing a combination of Newcastle Disease Virus (NDV), Everolimus, and Beclin-1. This combination was evaluated in a murine model, yielding findings that demonstrate enhanced tumor reduction and an improved immune response in comparison to monotherapeutic agents. The results substantiate the potential of this combination as a promising therapeutic strategy, thereby necessitating further clinical exploration.

Major Comments:

1. Although the study illustrates the efficacy of the NDV-Everolimus-Beclin-1 combination, there exists a paucity of mechanistic insight regarding the manner in which this combination affects autophagy and mTOR pathways. The manuscript would be considerably enhanced if the authors were to incorporate assays, such as Western blotting for autophagy and mTOR pathway markers, to directly elucidate the influence of Beclin-1 and Everolimus on these pathways within the context of NDV treatment.

2. The manuscript delineates multiple control and single-therapy groups; however, there exists a deficiency in the discourse concerning the efficacy of each monotherapy in isolation. For example, a more comprehensive analysis of the performance of NDV alone in comparison to Everolimus or Beclin-1 independently would yield a clearer baseline and enhance the comprehension of the synergistic effects present in the combination therapies.

3. The manuscript presently depends on caliper measurements for the evaluation of tumor growth, which may present limitations in accuracy for irregularly shaped tumors. The incorporation of in vivo imaging techniques, such as bioluminescence or fluorescence imaging, would produce more precise and reproducible measurements, thereby augmenting the credibility of tumor volume assessments.

4. Although the infiltration of immune cells, specifically CD8+ T cells, is referenced, the manuscript would benefit from a comprehensive histological analysis of tumor samples. IHC for CD8+ cells, along with markers indicative of apoptosis and proliferation, such as Ki-67, could yield valuable insights regarding the effects of treatment within the tumor microenvironment.

5. While the manuscript asserts enhanced survival rates, it fails to address the potential side effects or toxicity linked to the treatments, specifically regarding the triple combination. Incorporating a toxicity analysis, including parameters such as body weight monitoring, liver enzyme levels, or hematological profiles, is essential for evaluating the safety profile of this combination.

6. The immune response is assessed at a singular time point following treatment. Nevertheless, considering that immune responses progress over time, the authors ought to contemplate the measurement of cytokines and immune cell infiltration at multiple time intervals to comprehensively understand the dynamics of immune activation and suppression throughout the duration of treatment.

Minor Comments:

1. The introduction mentions potential mechanisms but lacks specific references, especially for the roles of NDV and Beclin-1 in cancer. Adding more recent and detailed citations will strengthen the background for the study.

2. The figure legends should more clearly explain the significance markers (e.g., *P < 0.05, **P < 0.01) to make it easier for readers to interpret the data. Additionally, the legends could specify which groups are being compared for each statistical marker.

3. Some figures (e.g., cytokine levels) show mean ± SD, while others do not specify error bars. Ensure that error bars are consistent and explicitly state in figure captions whether they represent standard deviation or standard error.

4. The abstract and introduction sections are dense with technical terms (e.g., "tumor-infiltrating CD8+ T cells," "immunogenic cell death"). Simplifying some language or briefly defining key terms would improve readability for a broader audience, particularly for interdisciplinary readers.

5. The methods section clarifies the titering method and specific titers used for NDV to ensure reproducibility. Adding information on how NDV was quantified (e.g., PFU/mL or EID50) would help other researchers replicate the experiment.

6. The ethics statement in the main text should consistently mention ethical approval details, including the institution’s name, approval code, and any specific animal welfare guidelines followed. Including these details enhances transparency regarding ethical compliance.

7. The manuscript does not address the potential impact of anesthesia on immune function. Some anesthetics, such as ketamine, can modulate immune responses. Briefly discussing or controlling for any possible confounding effects would strengthen the study's conclusions.

8. Add a concise explanation in the introduction of why these three agents were chosen for combination. For example, describe how NDV's oncolytic effect complements the immune-modulatory effects of Everolimus and Beclin-1 in targeting cervical cancer.

9. Some abbreviations (e.g., DAMPs, TAA) are not defined upon first mention, which may need to be clarified for readers unfamiliar with these terms. Ensure all abbreviations are fully defined the first time they appear.

10. The discussion briefly mentions the potential for clinical studies but could expand on specific challenges for clinical translation, such as optimizing dosing regimens or addressing potential toxicity in humans. This would give a balanced view of the translational feasibility of this combination therapy.

Reviewer #2: Manuscript # PONE-D-24-45377 entitled, “Boosting Immune Response against Cervical Cancer: A Combined Approach Using Oncolytic Virus and Targeted Therapies” evaluates the potential synergic effect of combination of Newcastle disease virus (NDV) along with Everolimus (EVE) and Beclin-1 (BEC) to improve immune reactions and decrease tumor development in an experimental model of HPV-related cervical cancer. To this end, using an HPV16 E6/E7- expressing TC-1 cells in C57BL/6 mice treated with NDV, EVE, BEC, or their combinations, E7-based-cytokine levels (IL-4, IFN-γ, IL-12), the infiltration of CD8+ T cells into tumors and Tumor growth was evaluated. Results indicated the synergic effect of the combination of NDV, EVE, and BEC in reduction of tumor growth by up to 70%, and elevating levels of E7-based IL-4, IFN-γ, IL-12 and CD8+ T cell infiltration as well as improved survival. Authors conclude this combination as a promising therapeutic approach.

Comments:

Application of NDV for cancer therapy of cancer patients in advance stages is reported decades ago. But the combinational approaches involving NDV and other chemotherapies is relatively new and thus this manuscript might report a relatively new approach for cancer therapy. Also the methodology and results of the present study are relatively clear and in accordance with the hypothesis of the study. But , in my opinion, the main and critical shortage of this submission is the discussion section! Indeed, surprisingly, when the main subject of this submission is evaluation of a new approach for inducing appropriate immune responses against tumor development in an experimental model of HPV-related cervical cancer, then we expect to see comparison of the “cytokine profile changes” and “reduction of the tumor growth” and “improvement of the survival” between the submitted study and that of the others but we don’t! That is, at least comparison of this study with that of the other similar studies with other approaches that measure the same parameters, at least with a few therapeutic vaccines that have specially used E7 as their target antigen. Of particular note, I strongly recommend the respected authors to compare their results with a recent study (Dorostkar et al, Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model, Infect Agent Cancer . 2021 Jan 26;16(1):7 ) and compare their IFN-γ and IL-4 levels as well as the anti-tumor activity. Moreover, I also recommend authors to compare their results for any possible parameter with the results of the recent following studies:

- Kazeruni et al, Newcastle disease virus enhances the antitumor efficacy of Doxorubicin in a cervical cancer mouse model, BMC Cancer . 2024 Oct 10;24(1):1253.

- Lee S et al, mRNA-HPV vaccine encoding E6 and E7 improves therapeutic potential for HPV-mediated cancers via subcutaneous immunization. J Med Virol. 2023 Dec;95(12):e29309. doi: 10.1002/jmv.29309.

- Ramos da Silva J et al, Single immunizations of self-amplifying or non-replicating mRNA-LNP vaccines control HPV-associated tumors in mice. Sci Transl Med. 2023 Mar 8;15(686):eabn3464.

- Mohapatra A et al, A sugar modified amphiphilic cationic nano-adjuvant ceased tumor immune suppression and rejuvenated peptide vaccine induced antitumor immunity in cervical cancer. Biomater Sci. 2023 Feb 28;11(5):1853-1866.

- Qi W et al, A novel multi-epitope vaccine of HPV16 E5E6E7 oncoprotein delivered by HBc VLPs induced efficient prophylactic and therapeutic antitumor immunity in tumor mice model. Vaccine. 2022 Dec 12;40(52):7693-7702.

_ Please clearly introduce groups of mice and what they have been immunized with (possibly as atable, please).

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Reviewer #1: Yes: Hossein Khorramdelazad

Reviewer #2: Yes: Farzin ROOHVAND

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<p>Boosting Immune Response Against Cervical Cancer: A Combined Approach Using Oncolytic Virus and Targeted Therapies

PONE-D-24-45377R1

Dear Dr. Shenagari,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Michael C Burger, M.D.

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Authors have addressed my comments in their revised version and thus I suggest acceptance of the manuscript.

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Reviewer #1: No

Reviewer #2: No

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