Review Reports - The Battle Against Pertussis: Discovery of Endogenous Human Proteins and Peptides as Toxin-Inhibitors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review tackles knowledge gap in utilizing Endogenous Proteins as Toxin-Inhibitors, with pertussis toxin as an example. The topic is timely with many recent research reports appearing in the field.

ADP ribosylating proteins’, and pertussis toxin’s in particular, significance is a highly covered topic. Thus, it is well that the manuscript has a well-defined scope, not to cover PT too much in general. On the other hand, pertussis has been outlined as the main focus, but the review at times makes more general claims to other diseases and toxins. I would encourage further narrow down these narratives even further, to improve the readability and conciseness of the review, and increase the impact of the review to address pertussis as a problem. Some examples:

- approach the introduction strictly from pertussis point of view. E.g., “Infections with bacteria” line 41 is very general and abstract. I think for clarity, better to focus on one pathogen to avoid too simplified narratives. This will shape and simplify narrative throughout introduction. Chapter 2 could just as well be the starting chapter.

- The above will aid to discuss antibiotic-resistance also from a more concrete angle: what is the situation specifically from B.pert point of view, and how important alternative treatments would be for this pathogen. More up to date references should e covered (than mattoo 2005) to discuss macrolide resistant strain development, in China the situation is worrisome, in Europe not yet so much.

- Figure 1 and 2 should be combined to be pertussis specific only

Although the review is about endogenous protein or peptide, also monoclonal antibodies to PT have been developed and proven to be protective in animal models (e.g., mice and baboons). Comprehensive comparison of advantages and disadvantages between the two approaches would be valuable for the readers. Current (lie 71-73) discussion is rather limited.

Abstract:

Lines 7-9 and 12-4 could be combined. Since the title and rest of abstract has been set as pertussis, the beginning of abstract can be more specific and only discuss b.pertussis.

Please specify Gai

“Despite the vaccination increasing case numbers were reported globally.” Not a very informative sentence, give decades, time spans.

“Thereby cellular responses and the characteristic clinical symptoms of the corresponding diseases are caused.” -> these molecular interactions cause cellular responses... or similar.

“The identification of human peptides/proteins as potent PT inhibitors such as defensins and a1-antitrypsin” -> The identification of human peptides/proteins, such as

defensins and a1-antitrypsin as potent PT inhibitors... right?

“should pave the way towards” -> Are promising candidates?

Introduction:

Line 36-37 unpolished - Simply: despite high vaccination coverage, several outbreaks...

Line 44-46, in a review, there is space to describe what are the exotoxin classification types.

51-52, 61-62 repetitive

Line 74 please specify further

Line 75 thought should be

78-80 revise language

80-83 – “we and others” is referred to, yet only a single reference is provided

The transfer of PT from B. Pertussis to outside of the cell and the formation and folding of the protein could be described early in “chapter 2”, since early intervention of the treatment is desired. This will help rationalize later discussion point (e.g., 176-179).

The link between accumulation of cAMP and clinical symptoms should be briefly covered (line 125-126)

Chapter 3: The need for treatment in children too young to be vaccinated as the important group of interest should be highlighted. Has this need decreased with the introduction of vaccination programs during pregnancy? Will this need decrease with time as these vaccination programs become more common? Although cases have increased, have hospitalization of infants increased? -> This information will help discuss the relevance of new treatments.

Line 164-166, for pertussis, nobody really knows what in the end is the causative agent of the disease. PT obviously has a role, for adenylate cyclase toxin, tracheal cytotoxin and dermonecrotic toxin all cause bad destruction of the epithelia and immune cells. I would suggest alternative description; the matter is unfortunately not that simple.

L169-170 As the authors describe: “Endogenous proteins and peptides are the first line of defense and belong to the innate immunity”, the role of PT in colonization should be covered, since that would be (one of) the preventive mechanisms? This is a highly disputed topic.

References: The reference and citation style are not according to journal guidelines and have to be updated.

Comments on the Quality of English Language

Many abbreviations appear in the text only a few times, it would be advisable to consider if introducing them is really necessary.

no need for Bordetella abbreviation (throughout the text).

Avoid we/our: “We and others are committed to identify” -> the scientific community is committed. “Here, we have summarized the recent efforts”: This review summarizes the recent efforts... This also comes later in other sections of the review.

Author Response

We thank this reviewer for the time spent upon our behalf. The very valuable comments and suggestions were very helpful to significantly improve the quality our revised manuscript.

POINT-BY-POINT RESPONSES:

The changes made in the revised version of the manuscript are highlighted in yellow.

Thank this reviewer very much for this valuable comment. The separate aspects are addressed in detail below.

Some examples:

We thank this reviewer for this comment, and we appreciate this point of view very much. However, the aim of this introduction was to give first a broad, simplified narrative and then to get into more detail in the following chapters. This should ensure a proper introduction of the structure, function, and mode of action of AB-type protein toxins. However, we followed the suggestions and changed the introduction accordingly. We hope that now the overall concept and potential of endogenous proteins and peptides as novel inhibitors for bacterial toxins gets more clearly introduced.

Chapter 2 could just as well be the starting chapter.

We understand and value the suggestion. However, we prefer to have a short introduction previously to chapter 2 to introduce the overall concept of the approach of searching for endogenous toxin inhibitors in peptide libraries from human tissues or body fluids. This should allow a broader readership to understand the basic principles of the article. The basic principle is to exploit endogenous proteins and peptides as novel pharmacological strategies to treat toxin-mediated diseases, here in detail pertussis.

We appreciate the comment of this reviewer very much. According to the suggestions we included more recent literature for macrolide-resistant B. pertussis. Moreover, we added more information on the geographic observation and mechanism of action of macrolide-resistant B. pertussis strains. The following was added to the revised manuscript: “Especially in certain parts of China, between 70-100% of clinical isolates showed a macrolide-resistance due to a specific mutation, a 2047 A-to-G mutation within the 23S rRNA gene [32–34]. In contrast, outside of China, there have been only sporadic reports on the appearance of macrolide-resistant B. pertussis strains [32,34].”.

- Figure 1 and 2 should be combined to be pertussis specific only

Thank you for this suggestion, but as mentioned above, we would like to keep the structure of a broader introduction to introduce the overall concept to a broad readership.

Thank you for bringing this topic to our attention. The monoclonal humanized antibodies under evaluation for pertussis are Hu1B7 and Hu11E6. These antibodies are not considered as endogenous proteins as they are engineered proteins. Since we primarily focused on endogenous proteins, we did not include them so far. However, these antibodies were already discussed as treatment strategy for pertussis within a previous literature review of ours: Ernst 2022. This review already contains detailed information, including the advantages and disadvantages of these antibodies. Since this topic is already covered within this previously published article we added the following to our revised manuscript: “For pertussis treatment, the monoclonal antibodies under evaluation in mice and baboon studies are Hu1B7 and Hu11E6. More detailed information, as well as advantages and disadvantages of these antibodies were described in more detail elsewhere [7]”.

Abstract:

Lines 7-9 and 12-4 could be combined. Since the title and rest of abstract has been set as pertussis, the beginning of abstract can be more specific and only discuss b.pertussis.

We fully agree and adjusted this part according to the suggestion made by this reviewer. The wording is now as followed: “The life-threatening disease pertussis, also known as whooping cough, is caused by a complex interplay of several virulence factors produced by the bacterium Bordetella (B.) pertussis. These includes the AB-type protein toxin pertussis toxin (PT), the main causative agent of pertussis. After infection with B. pertussis, PT is released and binds to its human target cells which internalize PT. The enzyme subunit of PT is then taken up into the cytosol where it catalyzes the ADP-ribosylation of the a-subunit of inhibitory GTP-binding proteins from the GaI type. This ultimately leads to the development of the characteristic clinical symptoms associated with pertussis.”.

Please specify Gai

Thank you for this suggestion. Specification was added to lines 11-12.

“Despite the vaccination increasing case numbers were reported globally.” Not a very informative sentence, give decades, time spans.

Thank you for highlighting this issue. The time span was added as indicated in line 15.

“Thereby cellular responses and the characteristic clinical symptoms of the corresponding diseases are caused.” -> these molecular interactions cause cellular responses... or similar.

We agree and deleted this part to streamline the abstract.

“The identification of human peptides/proteins as potent PT inhibitors such as defensins and a1-antitrypsin” -> The identification of human peptides/proteins, such as defensins and a1-antitrypsin as potent PT inhibitors... right?

“should pave the way towards” -> Are promising candidates?

We agree and modified this sentence now as followed: “The identification of endogenous peptides and proteins, e.g., defensins and a₁-antitrypsin, as potent inhibitors of PT pave the way towards the development of novel therapeutic options against pertussis.”.

Introduction:

Line 36-37 unpolished - Simply: despite high vaccination coverage, several outbreaks...

Thank you, we changed this accordingly.

Line 44-46, in a review, there is space to describe what are the exotoxin classification types.

We thank this reviewer for this very good suggestion and added the following part: “Bacterial exotoxins can be classified into three different types (Type I-III) [3,4]. Type I exotoxins cause signaling dysfunction via receptors on cell surfaces. Type II exotoxins damage host cell membranes. Type III exotoxins act intracellularly and are able to enter target cells and directly modify cell functions. The medically highly relevant AB-type protein toxins are type III exotoxins [3,4]”.

51-52, 61-62 repetitive

We see the point raised by this reviewer but would like to keep this part, as we think it is helpful to fully understand why antibiotics need to be applied early. It also explains why antibiotics are only effective before AB-type toxins are released by the bacteria and taken up by the target cells. Understanding that upon release and uptake of the toxins the substrate modification and the clinical symptoms are triggered is essential. However, if this reviewer strongly disagrees, we will delete it, of course.

Line 74 please specify further

Thank you for this valuable comment. Accordingly, we added further specification to the text passage. Here, further clinical interventions for pertussis treatment that comprise the treatment of the disease symptoms were added: “Further clinical interventions are limited to supportive or intensive care to manage the disease symptoms. For example, in severe pertussis cases in infants limited success was reported for extra-corporeal membrane oxygenation or oscillatory ventilation [8,9].”.

Line 75 thought should be

We are sorry but we do not understand the intent of this comment.

78-80 revise language

We have checked the language and corrected it. We also corrected “body-own” for “body’s-own” throughout the revised manuscript.

80-83 – “we and others” is referred to, yet only a single reference is provided

Yes, this is correct. Here, our recent literature review from March 2026 is cited that summarizes all previously performed work on this topic by us or others.

We are sorry but we do not really understand the intent of this specific comment. Maybe, there is some misunderstanding. The first part of the comment is not in connection with the discussion point of lines 176-179. The inhibition of PT secretion from B. pertussis is not topic of this review article. None of the introduced endogenous proteins/peptides of this review article were tested or described to inhibit PT secretion from B. pertussis. Here, only proteins/peptides are discussed that either inhibit the fully formed PT-holotoxin or its enzyme subunit PTS1.

The link between accumulation of cAMP and clinical symptoms should be briefly covered (line 125-126)

We thank the reviewer for bringing to our attention that additional information would be useful. Therefore, we added the following to the test passage: “Elevated cAMP levels are cell type dependent and influence two down-stream signaling cascades, protein kinase A (PKA) and the exchange proteins directly activated by cAMP (EPAC) [7,27]. PKA and EPAC signaling are involved in diverse immune and metabolic pathways, causing systemic immunosuppression, lymphocytosis, and metabolic disruptions [27].”.

Chapter 3:

The need for treatment in children too young to be vaccinated as the important group of interest should be highlighted. Has this need decreased with the introduction of vaccination programs during pregnancy? Will this need decrease with time as these vaccination programs become more common? Although cases have increased, have hospitalization of infants increased? -> This information will help discuss the relevance of new treatments.

We appreciate the questions raised by this reviewer and addressed them in a brief statement within the revised manuscript. The following was added: “In general, the vaccination is an essential strategy to prevent pertussis. However, infants (<6 months) are a population group at high risk for severe pertussis, since they are unimmunized or only partially immunized [2]. Here, the importance of maternal immunization vaccination programs for pertussis should be communicated early during pregnancy, to protect newborns from pertussis with maternal antibodies [2]. At the same time, for population groups at high risk, effective treatment strategies are urgently required. Consequently, novel targeted strategies that address the treatment gap which opened due to evolved B. pertussis strains and to protect vulnerable groups at high risk for severe pertussis are urgently required.”.

We absolutely agree. Of course, the interplay of several virulence factors mediates pertussis disease. However, PT is one of the most important virulence factors of B. pertussis and responsible for many key events of pertussis pathology (summarized by Scanlon K, Skerry C, Carbonetti N. Adv Exp Med Biol. 2019;1183:35-51. doi: 10.1007/5584_2019_403.). We have added this reference to the revised version of the manuscript (line 115).

To our best of knowledge, studies on the relevance of defensins or antitrypsin on PT during the colonialization of B. pertussis in humans are not available so far. Therefore, novel approaches for the prevention of B. pertussis colonialization were not covered within this manuscript. However, this is a very valid and interesting aspect for further studies involving proteins and peptides as novel treatment strategies for pertussis in the future.

References: The reference and citation style are not according to journal guidelines and have to be updated.

Thank you for the comment. We corrected the citation style to the MDPI (Multidisciplinary Digital Publishing Institute) citation style of the journal.

Language comments

Many abbreviations appear in the text only a few times, it would be advisable to consider if introducing them is really necessary.

We understand the concern of this reviewer. The abbreviations which were introduced will be kept for sake of the reading flow. We explain the abbreviations when used the first time in the text and then consistently use only the abbreviations, according to the editorial style of this journal. However, if this reviewer strongly disagrees, we will delete the abbreviations.

no need for Bordetella abbreviation (throughout the text).

We appreciate the comment. We have checked again the literature and found that the common abbreviation for “Bordetella pertussis” is “B. pertussis”. Therefore, we would like to keep this abbreviation as it appears to us to be well-known. Of course, if the reviewer insists, we will delete the abbreviation for Bordetella.

We appreciate the feedback on the language and changed the sections accordingly.

Overall, we are convinced that our manuscript has significantly improved by the changes made following the valuable suggestions and comments of the reviewers. We hope that the revised version of our manuscript is now acceptable for publication in Toxins.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The current review entitled “The Battle Against Pertussis: Exploitation of Endogenous Proteins as Toxin-Inhibitors” summarizes the recent efforts in the identification and characterization of human body own proteins and peptides that inhibit PT. The identification of human peptides/proteins as potent PT inhibitors such as defensins and α1-antitrypsin should pave the way towards novel therapeutic strategies against this severe pertussis disease. I think this is a broad name not specific to what is included in the review. I have also some comments:

The abstract is not structured and looks like an introduction not abstract to all included in the review.
Self-citation is considered as the authors mentioned we for references (36-39) many times
Many paragraph and facts are mentioned without cited references (the whole manuscript needs meticulous revision)
Many paragraphs are mentioned without specific aim, just repetition.
Many reference are very old and need to be updated
English language needs revision.
Other comments in the attached manuscript.

Best regards,

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Dear authors and editor,

Many paragraphs are mentioned without specific aim, just repetition.
English language needs revision.

Best regards,

Author Response

We thank this reviewer for the time spent upon our behalf. The very valuable comments and suggestions were very helpful to significantly improve the quality our revised manuscript.

POINT-BY-POINT RESPONSES:

The changes made in the revised version of the manuscript are highlighted in yellow.

I think this is a broad name not specific to what is included in the review.

We appreciate the suggestion made by this reviewer. Therefore, we have adjusted the title of the revised manuscript to: “The Battle Against Pertussis: Discovery of Endogenous human Proteins and peptides as Toxin-Inhibitors”.

I have also some comments:

The abstract is not structured and looks like an introduction not abstract to all included in the review.

We fully agree and modified the abstract accordingly, also on the basis of the suggestions made by reviewer 1.

Self-citation is considered as the authors mentioned we for references (36-39) many times

We see this point and changed it accordingly throughout the manuscript.

Many paragraph and facts are mentioned without cited references (the whole manuscript needs meticulous revision)

We thank this reviewer very much for this comment. The comments made by this reviewer in the provided pdf version of our manuscript are addressed in detail in the revised manuscript, as described in the points below.

Many paragraphs are mentioned without specific aim, just repetition.

We tried to improve this throughout the manuscript wherever we had the impression of repetition.

Many reference are very old and need to be updated

We have checked the references that were marked by the reviewer in the attached pdf version of the manuscript. Here, primary literature was marked as “old”. E.g., Stein et al. were the first to solve the crystal structure of PT. We feel that this key publication should be cited to give credit to researchers that did this groundbreaking initial work in pertussis research.

Other comments in the attached manuscript:

Line 29: Are not both the same?

We tried to make this point clearer now. Pertussis toxin belongs to the group of AB-type protein toxins but there are also other toxins that are AB-type protein toxins. Therefore, pertussis toxin and AB-type protein toxin are no synonyms.

Line 35-39: Please cite your references

We appreciate this comment. We have checked the corresponding lines within the manuscript. Here, already two references were cited. However, if this reviewer insists, we can add further references.

Line 49-55: Please cite your references

Done.

Line 61-68: Please cite your references

Done.

Line 109: Please update these references

Here, primary literature is cited. We believe that the reference is correct. Please, see the comment above.

Line 140-142: Do you have an agreement from these authors or it is self constructed figure?

All figures of this manuscript, as indicated, are self-constructed figures using BioRender. This point was also officially sorted out before with the editorial office of the journal.

Line 162-167: Can you summarize these strategies?

We thank this reviewer for this comment. Here, we aim to highlight that novel strategies are required. This review article focuses on endogenous proteins and peptides as novel strategies (line 171-191). In more, detail human defensins and a₁-antitrypsin are described (chapter 4).

Line 169-179: What is the aim from this paragraph?

We appreciate the comment. The aim of this paragraph (189-203) is to highlight the potential of endogenous proteins and peptides as novel treatment strategies for toxin-mediated diseases. Since as part of the innate immunity antimicrobial peptides have developed it is likely that the human proteome/peptidome also contains proteins/peptides with anti-toxin activity. Also, the complexity of the human proteome/peptidome contributes to a high probability to identify proteins/peptides with anti-toxin activity

Line 255-281: Please cite your references

Thank you for this comment. As already clearly mentioned in the manuscript, this paragraph summarizes the work from Kling et al., 2021, 2023. This is clearly stated line 257-258 and throughout table 1. However, for further clarity we slightly rephrased the sentence and specifically mention Kling and colleagues (line 269-270) in the revised manuscript.

Line 398-400: Conclusion should not include reference

We very much appreciate this comment. We have checked again the author instruction guidelines and could not find a section stating that the conclusion should not include any references. Moreover, we have crosschecked within recently published review articles of the journal Toxins that review articles with a conclusion indeed include references. Therefore, we prefer to keep the citations made in the conclusion.

Line 531-543: Could be self citation as the authors mentioned we for these references many times

Thank you for the comment. We are aware of this issue as this manuscript is based on own research. However, we also included additional references where it was possible and adequate in the revised version of the manuscript. We hope this will address this issue.

Language comments:

Many paragraphs are mentioned without specific aim, just repetition.

We thank this reviewer for this comment and have made specific adjustments also in accordance with the comments made by reviewer 1 and 3.

English language needs revision.

We agree and have made corresponding adjustments throughout the revised manuscript as we aim to submit a clear and comprehensive review.

Reviewer 3 Report

Comments and Suggestions for Authors

1)The manuscript is generally understandable but contains multiple grammatical issues, awkward phrasing, and typographical errors (e.g., “thereby cellular responses… are caused,” “body-own proteins”).

2)The review summarizes recent work on endogenous inhibitors of pertussis toxin (PT), but the novel contribution relative to existing reviews is not clearly articulated.

3)Although therapeutic potential is emphasized, clinical applicability is underdeveloped.

4)The manuscript structure is logical, but some sections are overly long.

5)Table 1 is useful but could be improved: Formatting inconsistencies (alignment, spacing) Mechanism column is sometimes vague (“not tested yet”) Consider summarizing key takeaways below the table

Author Response

We thank this reviewer for the time spent upon our behalf. The very valuable comments and suggestions were very helpful to significantly improve the quality our revised manuscript.

POINT-BY-POINT RESPONSES:

The changes made in the revised version of the manuscript are highlighted in yellow.

1) The manuscript is generally understandable but contains multiple grammatical issues, awkward phrasing, and typographical errors (e.g., “thereby cellular responses… are caused,” “body-own proteins”).

We thank this reviewer for this valuable comment. We have corrected these errors throughout the revised manuscript.

2) The review summarizes recent work on endogenous inhibitors of pertussis toxin (PT), but the novel contribution relative to existing reviews is not clearly articulated.

We are sorry that this did not became clear. We added this information now clearer as follows: The description of the key contribution was added, line 32-35: “This review highlights the potential of endogenous human proteins and peptides, in specific a-defensins and a₁-antitrypsin, which directly and specifically target and neutralize pertussis toxin. This should offer novel pharmacological strategies to treat pertussis in the future.”.

3) Although therapeutic potential is emphasized, clinical applicability is underdeveloped.

Thank you for the statement on the current state of endogenous proteins/peptides for the treatment of pertussis. This is highlighted in the review as no clinical data from humans are included. Here, results from basic research are summarized, as well as first in vivo data from a B. pertussis mouse model and cytotoxicity testing from zebrafish embryos.

4) The manuscript structure is logical, but some sections are overly long.

Thank you for the comment. We shortened some sections.

5) Table 1 is useful but could be improved: Formatting inconsistencies (alignment, spacing) Mechanism column is sometimes vague (“not tested yet”) Consider summarizing key takeaways below the table

We thank this reviewer very much for recognizing this mistake. During formatting performed by the journal, alignment and spacing was changed. We fixed the errors now. However, we do not understand why the statement “not tested yet” is vague. For some peptides, the mode of inhibition was not evaluated and therefore “not tested yet” is the most accurate or concrete statement that can be given here. The key findings of the table are summarized in figure 3.

As requested by the editorial office, we added the following short section “Key Contribution” on the title page:

“This review highlights the potential of endogenous human proteins and peptides, in specific a-defensins and a₁-antitrypsin, which directly and specifically target and neutralize pertussis toxin. This should offer novel pharmacological strategies to treat pertussis in the future.”

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author's have considered the comments relevant to scientific discussion, and left most comments related to style and structure, a freedom which I am happy to leave at the author's decision.

Author Response

The author's have considered the comments relevant to scientific discussion, and left most comments related to style and structure, a freedom which I am happy to leave at the author's decision.

We thank this reviewer for the friendly and valuable feedback and all comments which helped to improve this manuscript during the revision.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The revised version is good, just the abstract could include more about what you have done and concluded. also, conclusion should not include references, you can add this part in the discussion. the conclusion should involve what you have concluded from your research or readings.

Best regards,

Comments on the Quality of English Language

Dear editor,

English language in the revised version is good.

Best regards,

Author Response

We thank this reviewer for these comments. To include more about the content of the manuscript within the abstract, we have added the following sentence to the abstract: “PT inhibiting peptides were found by unbiased screening of peptide libraries from human hemofiltrate or hypothesis driven evaluation and PT neutralizing mechanisms were discovered in cell-based approaches.”.

Moreover, we moved the following sentences from the conclusion to the end of chapter 4.2: “The findings with PT provided further prove-of-concept that the generation of peptides based on endogenous human protein precursors can result in the generation of peptides with anti-toxin activity, which has a scientific and clinical impact for various protein toxins from other medically highly relevant bacteria, e.g., C. difficile, which are also neutralized by human peptides and proteins [73,74].”.

Therefore, there a no references in the conclusions in the revised manuscript.

English language in the revised version is good.

We thank this reviewer very much for this comment.

Reviewer 3 Report

Comments and Suggestions for Authors

none

Author Response

Comments and Suggestions for Authors: none

We thank this reviewer for the time spent on our behalf and the valuable comments during the review process.