Development of High-Throughput Serum Bactericidal Assays for Bordetella pertussis to Evaluate BPZE1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript by Goldstein et al. describes a serum bactericidal assay

to address comparison of BPZE1 (live attenuated pertussis vaccine) with current 

and future vaccines. An important issue is the increase in PRN-negative strains causing

 infection/disease worldwide. The study includes extensive operational controls to justify 

the comparisons to previously employed assays. This presentation represents an appropriate

 next step in the process of validating the effectiveness and use of BPZE1 and    
other products to be developed. 

 

Comments on the Quality of English Language

The manuscript is well written, but could be improved with review of hyphenation and punctuation

 

 

 

 

 

 

Author Response

Thank you for the thoughtful review. We have reviewed and addressed your comment as follows:

Comment 1: The manuscript is well written, but could be improve with review of hyphenation and punctuation:

Reply 1: We have reviewed the document and addressed all punctuation and grammar findings.

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors deliver a very sound article on the new design of the serum bactericidal assay. The assay is specifically tailored to address the problems related to vaccines against Bordetella pertussis. The B. pertussis infections have been under limited control by the existing vaccines. However, the efficacy of these vaccines is not sufficient, as they cannot provide effective ways of hampering transmission of the pathogen. Therefore, mere neutralization of the pertussis toxin is not enough. Therefore, pertussis research aims at the vaccine compositions that would induce bactericidal antibodies.

The main objective of this solid work is the development and optimization of the new approach to a high-throughput testing of the bactericidal activities of specific antibodies in the presence of complement. 

The study provides a detailed analysis of the technicalities of the test design and performance. The optimizations (including complement and antibodies selection, B. pertussis test strain selection, plate layout, etc.) were meticulously designed and justified.

As it is not a research paper per se, it deals with the important methodological aspect of the measurement of bactericidal activities of antibodies in the presence of human complement.  It presents an overall design and validation of the method, along its implementation to a specific context of BPZE1 vaccine with direct practical applications. It constitutes a valuable addition to the pool of B. pertussis vaccine - related knowledge and is well suited for the publication in Vaccines.

Regarding the technicalities of the test design, what I have not found elaborated upon in the article is how the actual counting of the colonies was done. The Authors claim that the approach is of a high-throughput, but there is no description of the final measuring step (that is counting the colonies). Is it fully automatic and/or supervised? And lastly what tools (programming tools for that matter) were used for identifying and enumerating the colonies in the wells of the plate. I would gladly see a bit of a discussion on the possible alternatives to the Zeiss AxioLab 4 and image analysis system (AxioSystem). This constitutes my recommendation to be used at the Authors' discretion.  

In my view, the article can be forwarded for publication.

Author Response

Thank you for the thoughtful and detailed review. We agree with your comment and have addressed it below.

Comment: Regarding the technicalities of the test design, what I have not found elaborated upon in the article is how the actual counting of the colonies was done. The Authors claim that the approach is of a high-throughput, but there is no description of the final measuring step (that is counting the colonies). Is it fully automatic and/or supervised? And lastly what tools (programming tools for that matter) were used for identifying and enumerating the colonies in the wells of the plate. I would gladly see a bit of a discussion on the possible alternatives to the Zeiss AxioLab 4 and image analysis system (AxioSystem)

Reply: Thank you for your review of our manuscript. We are pleased to provide additional information regarding your comment. You will find three additional sections (sections 2.4.1, 2.4.2, and 2.4.3, lines 162-193) which add additional clarity to the technical aspects of the method discussed.

Reviewer 3 Report

Comments and Suggestions for Authors

Against the backdrop of the global COVID-19 pandemic, the prevalence of PRN-negative Bordetella pertussis strains represents a significant scientific issue in the fields of veterinary and public health. This article systematically develops and validates a high-throughput serum bactericidal assay (SBA) targeting both PRN+ and PRN- B. pertussis strains, addressing a key challenge in current pertussis vaccine evaluation. This work holds important clinical and public health significance. The study design is rigorous, the experimental data are substantial, and the results validate the accuracy, linearity, specificity, and robustness of the method, providing a critical technical approach for evaluating the clinical immunogenicity of live attenuated pertussis vaccines. The article is well-written, with fluent language and comprehensive references, making it suitable for publication in the journal Vaccines. The topic and overall quality of this research article are high, but a few points require clarification or revision.

• P4L143: Is the notation "120+/-5" a typo? Should it be "120 ± 5"? Please check the entire manuscript for similar notations.

• P4L151: Can the resolution of Figure 1 be further improved?

• P4L161: The labeling "2.4" duplicates the "2.4" found on P3L33.

• P5L172: The resolution of Figure 2 is too low.

• P8L340: Sample 2, used to determine the Lower Limit of Precision (LLP), had only 5 reportable titers out of 12 determinations (i.e., less than 50% valid data), which seems somewhat insufficient. Another sample (Sample 1) also could not provide valid support. Although the authors state that further validation with more samples is needed, it is recommended that the authors more explicitly acknowledge the uncertainty of the current LLP in the discussion and confirm it using more low-titer samples before Phase 3 clinical trials.

• P9L358: The meaning of "N" in Table 1 is not clearly explained in the table footnote. There is an inconsistency in the data for the PRN-SBA between the main text (217) and Table 1 (216). Is this a typographical error, or was an outlier excluded? Please verify.

• P14L471: The authors compared the new method (agar-overlay) with the old method (tilt-plate) and discussed the main differences between the two methods in the discussion. The authors should more specifically point out the fundamental shortcomings of the old method. Why was the old method widely used despite these shortcomings? The primary apparent difference between the new and old methods is the tilt-plate vs. agar-overlay technique. Why does this change confer a fundamental advantage to the new method? Please explain the core rationale in the introduction or discussion.

• P19L619: The authorship of reference 5 is not formatted correctly. It is recommended to re-check the completeness and correctness of all references.

Author Response

Dear Reviewer 3: We thank you for your thorough review and agree with all of your comments and suggestions. Please find the details below:

P4L143: Is the notation "120 +/- 5" a typo? Should it be "120 ± 5"? Please check the entire manuscript for similar notations.

Reply: Thank you for pointing this out. We have changed the “+/-“ to “±” throughout the document.

P4L151: Can the resolution of Figure 1 be further improved?

Comment: We have updated Figure 1

 

P4L161: The labeling "2.4" duplicates the "2.4" found on P3L33.

Reply: We have updated the sections accordingly.

 

P5L172: The resolution of Figure 2 is too low.

Reply: We have addressed this now with a higher resolution image.

 

P8L340: Sample 2, used to determine the Lower Limit of Precision (LLP), had only 5 reportable titers out of 12 determinations (i.e., less than 50% valid data), which seems somewhat insufficient. Another sample (Sample 1) also could not provide valid support. Although the authors state that further validation with more samples is needed, it is recommended that the authors more explicitly acknowledge the uncertainty of the current LLP in the discussion and confirm it using more low-titer samples before Phase 3 clinical trials.

Reply: This was a very astute observation which we are glad to clarify. We have added a paragraph in the discussion to discuss this detail. It can be found on P17L573

 

P9L358: The meaning of "N" in Table 1 is not clearly explained in the table footnote. There is an inconsistency in the data for the PRN-SBA between the main text (217) and Table 1 (216). Is this a typographical error, or was an outlier excluded? Please verify.

Reply: Thank you for catching this. The number should be 216 in both places and has been adjusted.  A footnote has been added to the table explaining "N".

P14L471: The authors compared the new method (agar-overlay) with the old method (tilt-plate) and discussed the main differences between the two methods in the discussion. The authors should more specifically point out the fundamental shortcomings of the old method. Why was the old method widely used despite these shortcomings? The primary apparent difference between the new and old methods is the tilt-plate vs. agar-overlay technique. Why does this change confer a fundamental advantage to the new method? Please explain the core rationale in the introduction or discussion.

Reply: We have added additional discussion about this in the discussion section.  P17L549

P19L619: The authorship of reference 5 is not formatted correctly. It is recommended to re-check the completeness and correctness of all references.

Reply: We have re-checked all the references and corrected them accordingly.

Author Response File: Author Response.docx