PLOS Pathogens

Dear Dr. Agrawal,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 28 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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* A letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

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If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Anne Jamet

Section Editor

PLOS Pathogens

Anne Jamet

Section Editor

PLOS Pathogens

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Babita Agrawal, Shanika Werellagama, Raj S Patel, Nancy Gupta, Satish vedi, Diana Duque, Jegarubee Bavananthasivam, Rakesh Kumar, and Anh Tran. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

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If you did not receive any funding for this study, please simply state: u201cThe authors received no specific funding for this work.u201d

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In the current study by Werellagama et al., the authors have demonstrated the potential of heat killed Caulobacter crescentus (HKCC), a freshwater, non-pathogenic bacterium as an innate immunomodulatory agent. They showed that the HKCC treatment in respriratory infection animal models can induce robust localized and systemic mucosal immunity through comprehensive set of immunological and histopathological assays and imaging. They have shown that the HKCC treatment also bolsters adaptive immunity especially in viral infections like flu and SARS-CoV2. In summary, their data suggest both preventive and to some degree post-infection control of respiratory pathogens more effectively by boosting innate and adaptive immune responses. The study is clinically relevant and demonstrate the potential of immunomodulatory agents to boost mucosal immunity for better infection control.

Reviewer #2: Werellagama et al investigated a host-directed immunotherapeutic strategy against respiratory bacterial (Mycobacterium avium, Mav) and viral (SARS-CoV-2 and influenza) infections in animal models. This strategy used heat-killed Caulobacter crescentus (HKCC) as an innate immune modulator. They found that intranasal administration with HKCC stimulates innate lymphoid cells and neutrophils while enhances antigen-specific antibody production, resulting in reductions in both bacterial and viral loads and significantly milder disease progression. The authors indicated that HKCC may offer an effective, ready-to-use strategy to enhance the host defence against a broad range of bacterial and viral respiratory pathogens, warranting future clinical trials. There are, however, some major issues to be carefully addressed.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: First, it is unclear if HKCC can be generalised for clinical development against broad respiratory bacterial and viral infections. The data in figure one is unclear on how these time points were selected for intranasal administration. Since animals that received HKCC intranasally 21 and 8 days before challenge, along with untreated-infected controls, showed a more inflammatory profile with increased type 1 ILCs than ones received HKCC intranasally 8 and 1 days before challenge, this finding indicates a complex situation for clinical use of such innate immune modulator. The timing, dose and frequency for real world use would be hard to determine. It is, therefore, necessary to solve the underlying mechanism for a consistent innate immune modulatory effect.

Second, the authors did not address a critical issue on the impact of host pre-existing immunity. Since HKCC is common, the authors may need to determine if host pre-existing immunity to HKCC would have a major influence on the efficacy of such an intervention. To this end, if the HKCC intranasal administration induces anti-HKCC immunity, it is necessary to determine if the induced immunity would prevent the future efficacy of the same product.

Third, during the experiments, the authors used three infection models. They, however, did not provide convincing data if these models engaged the same mechanism of protection. This is a critical issue because it is known that some people may generate worse cytokine storms than others after SARS-CoV-2 infection. If HKCC generates worse inflammatory responses in these individuals, the safety evaluation should be done in more relevant clinical model.

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Rationale for bacterial choice and novelty:

The rationale for selecting Caulobacter crescentus over other bacterial genera is not sufficiently justified beyond its non-pathogenic nature. In principle, bacteria that more closely mimic respiratory pathogens might have been more appropriate for inducing relevant mucosal immune responses. Moreover, while the concept is interesting, it is not entirely novel. Previous studies have demonstrated that bacteria or bacterial extracts (e.g., OM-85, Lactobacillus species) can induce mucosal immunity in the respiratory tract and gut. These studies should be incorporated into the Introduction or Background, with a clearer discussion of how the current work advances or fills gaps in existing knowledge. In addition, the study lacks comparative controls using bacteria from other genera or established immunomodulatory agents. Including one or two well-characterized adjuvants would have strengthened the study design and contextualized the observed effects of HKCC.

Relevance to immunocompromised hosts:

Although HKCC treatment markedly improves disease outcomes in the Mycobacterium avium infection model, it remains unclear whether such robust immune induction would occur in immunocompromised settings, where mycobacterial infections are most clinically relevant. This limitation should be acknowledged, and the potential variability of HKCC efficacy in immunocompromised hosts discussed.

Durability and limitations of preventive and therapeutic efficacy:

The authors propose HKCC as both a preventive and adjunct therapeutic approach for respiratory infections. A major concern for the preventive strategy is the durability of the induced innate immune responses. The data clearly show that HKCC administration 24 hours prior to infection (8–1 day scheme) yields superior protection compared to the longer interval (21–8 day scheme), suggesting that the immune response may be relatively short-lived. This could represent a significant hurdle for preventive applications. Furthermore, the post-infection treatment approach appears less effective, as evidenced by comparatively modest disease control (e.g., body weight changes and viral titers in treated groups). These limitations should be explicitly discussed, as they warrant further investigation and optimization.

Variability in experimental design across infection models:

The treatment and infection schedules differ substantially across the three infection models used in the study. A clearer rationale for these variable experimental designs is needed, along with an explanation of how these differences impact data interpretation and cross-model comparisons.

Reviewer #2: Fig 1, is the protective efficacy dose-dependent? Why was only 5x105 PFU used for this experiment? The scale bar for each H.E. image is missing.

Fig 2, was there any statistical significance for T-Bet or other parameters measured?

Fig 3, did the lung IgA show any protective potency as compared with IgG?

Fig 4 & 5, did innate lymphoid cells and neutrophils correlate with protection?

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PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes: Swati Jain

Reviewer #2: No

Figure resubmission:

Reproducibility:

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Dear Dr. Agrawal,

We are pleased to inform you that your manuscript 'Host-directed Broad-spectrum Immunotherapeutic Strategy for Respiratory Infections: Heat-killed Caulobacter crescentus (HKCC) as an Innate-immune based Biotherapeutic/Postbiotic' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Anne Jamet

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors have thoroughly addressed the reviewers’ comments and incorporated the suggested revisions. Importantly, they have also added a clear discussion of the study’s limitations, which strengthens the overall rigor and transparency of the manuscript.

Reviewer #2: This study report a strategy using heat-killed Caulobacter crescentus (HKCC) as a potential innate immune modulator.

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PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: Yes: Swati Jain

Reviewer #2: No

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)