Development and Immune Efficacy Evaluation of Two Live Triple-Gene-Deleted Vaccine Candidates Against Bovine Herpesvirus Type 1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors provide strong justification for this investigation and report.  Study design and methods are effectively described.  There are some questions on details and there is an opportunity to improve the clarity of writing in a few areas. Overall, this is a well-prepared report with substantial data to support conclusions.

Specific comments,

Line 110, Figure 1 is lacking a title, legend or description.

Line 146, Safety evaluation, as written it is not clear this section should specify the rabbits from the inoculation of mutant virus injection. As written the methods are not clear.

Line 156, similarly when the authors describe the treatment groups, it remains unclear what is being referenced.  For instance, in the first line, this should include description of the subjects (rabbits) that are blocked into groups and the treatments that each block received.    

Line 230, the use of the word extremely is redundant.  The data were significantly different.

Line 239, same comment.   

Author Response

Comments 1 :Line 110, Figure 1 is lacking a title, legend or description.

Response 1: We apologize for this omission. We have now added a complete title, legend, and description to Figure 1. The revised figure caption reads: Figure 1. Schematic diagram of the two-step recombination for BoHV-1 BAC-ΔTK. This has been updated in the revised manuscript (line 111).

Comments 2 : Line 146, Safety evaluation, as written it is not clear this section should specify the rabbits from the inoculation of mutant virus injection. As written the methods are not clear.

Response 2: We agree with the reviewer that the description of the safety evaluation was ambiguous. To address this, we have now included a detailed table 1(see line 157) that clearly presents the grouping of rabbits and the specific treatments each group received, including the inoculation of mutant virus. The safety evaluation methods have been rewritten accordingly in the revised manuscript (line 146 onwards), referring to the table. We believe this makes the experimental design and the safety assessment procedures completely clear.

Comments 3:Line 156, similarly when the authors describe the treatment groups, it remains unclear what is being referenced. For instance, in the first line, this should include description of the subjects (rabbits) that are blocked into groups and the treatments that each block received.

Response 3: We thank the reviewer for this valuable suggestion. To clarify the description of the treatment groups, we have now summarized the detailed grouping information, including the subjects (rabbits) and the specific treatment each group received, in a new table(line 168).

Comments 4:Line 230, the use of the word extremely is redundant. The data were significantly different.

Response 4: We agree with the reviewer that the word “extremely” is unnecessary. We have deleted it from line 230 in the revised manuscript. The sentence now reads: “The data were significantly different between the two groups.” Thank you for improving the clarity of our language.

Comments 5:Line 239, same comment.

Response 5:We thank the reviewer for the same suggestion. The word “extremely” has also been removed from line 239 in the revised manuscript. The revised sentence now reads: “The differences observed were statistically significant.” We appreciate the reviewer’s careful reading.

Reviewer 2 Report

Comments and Suggestions for Authors

Infectious bovine rhinotracheitis is a severe disease of cattle that can have devastating effects in affects farms. The infection causes a multitude of disease syndromes and currently there are significant problems internationally in the efforts to control it.

The authors rightly have made a tremendous effort to develop a novel vaccine, through a novel and hitherto unused methodological approach, in order to achieve effective control of this important problem. They are to be commended for the excellent manuscript that they submitted for evaluation.

The manuscript can stand as it is and can be published immediately.

However, some minor changes in the manuscript will make it read better and will further ameliorate its readability.

-Perhaps, instead of construction, to use ‘Development’?

-Fantastic idea to use rabbits instead of cattle for the evaluation of the developed vaccine. I suggest to include a paragraph in Discussion to explain the advantages of this approach.

-In M&M, please add a table to describe the experimental design of the study in a graphical mode.

-Also, please give details of manufacturers of all consumables and equipment used in the study.

-Please explain clearly the control procedures employed in this experimental setting. Perhaps, you can insert a new sub-section specifically for this purpose.

-In Results, please improve visualization by inserting additional graphs.

-In Discussion, please explain why safety of the new product in rabbits is much more important than in cattle.

-I suggest to divide the Discussion in subsections.

-Please add a new paragraph explaining a brief marketing plan for this novel product.

-Conclusions must be finished with a strong takeaway message.

 

 

Overall. Excellent manuscript for immediate acceptance after submitting the revised and improved version.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, the manuscript presents a competent study on two triple-gene deleted BoHV-1 vaccine candidates (∆T3: ∆TK-∆gE/gI; ∆g3: ∆gG-∆gE/gI), using a rabbit model for safety and efficacy evaluation. It demonstrates good molecular biology work and aligns with established practices in alphaherpesvirus vaccine development. However, it has several concerns that make this work not suitable for publication.

1. In the method, the authors indicate that the rabbits were challenged via intranasal inoculation with the "BoHV-1 BC01" strain. But the legend for Fig 8 clearly states that the histopathological images of the lung tissues are from after a "BoHV-1 HB06" challenge. I think this is a notable discrepancy that likely implies either extreme carelessness or, possibly, the recycling of histopathology images from a completely different study. The authors must provide a logical explaination to this mistake.
2. Section 2.7 and Fig. 6B  characterize viral loads as nucleic acid copies quantified by qPCR. However, Section 3.4.3 describes these results using "TCID50/mL. This conflation of genomic copies with live virus titers represents an error that must be carefully addressed.
3. The experimental timeline, which challenged the inactivated vaccine group only 7 days after their booster, is actually not enough for a peak secondary humoral response. I think this design could bias the results against the inactivated control and create an unfair advantage for the live vaccine candidates.
4. In the introduction, the authors critique current inactivated vaccines for their failure to induce robust cellular immunity. Despite using this as a primary rationale for developing their live-attenuated candidates, they fail to present cellular immunity data (e.g., IFN-γ levels, T-cell proliferation assays) to prove their candidates overcome this limitation.
5. The authors used commercial blocking ELISA kits for gE and gB antibody detection. However, since these assays are typically optimized for bovine sera, their application to rabbit samples without species-specific validation could raise concerns regarding the analytical specificity and sensitivity of the results in this host model. Please address this point.
6. I think that the authors' claim of vaccine dose-dependency at 42–49 dpi is scientifically invalid. These antibody titers likely reflect the anamnestic response to the massive viral challenge at 28 dpi rather than the vaccine itself.
7. The y-axis labels in Fig. 4A, 4C, and 6B, which denote viral shedding as "viral load (lg copies/mg)," are inconsistent with the method. Since nasal swabs were processed in 500 μL of PBS, using milligrams is inappropriate for a liquid suspension. This error suggests to me that axis templates from a different assay, likely involving tissue samples, were copied and pasted without the necessary adjustments for the current data.
8. Section 3.2 contains significant typographical errors regarding the reported viral peak titers. The values for rBoHV-1-Ag3 and the wild-type strain are presented inaccurately.
9. Fig. 7 clearly displays gross, macroscopic images of whole rabbit lungs. However, the figure legend describes it as "Post-challenge pulmonary histopathological changes". The actual histopathology (H&E staining) is shown in Fig. 8.  
10. The Materials and Methods section entirely omits any protocol for scoring or evaluating macroscopic gross lung pathology. The authors present gross pathology results in Fig. 7 without ever stating how the lungs were extracted, preserved, or evaluated for macroscopic lesions.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has improved greatly with the changes made.
Perhaps the authors can add a passage in the Conclusions regarding the potential for further development and commercialization.
Then the manuscript will be 100% ready for acceptance.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have provided the improved version of the manuscript. However, there is a major concern that I think should be clearly addressed.

The authors admitted to conflating TCID50 with nucleic acid copies. To "fix" this, they simply swapped the unit labels in the text and axes. However, after carefully looking at the actual values, in Section 3.4.3, they now state the viral load peaked at approximately 10^3.9 copies/mL. For a qPCR assay detecting viral shedding from nasal swabs at the peak of an active alphaherpesvirus infection, 10^3.9 (less than 8,000) copies/mL is exceptionally low. This is actually at the borderline background noise. True genomic copy numbers during active shedding should be several logs higher (e.g., 10^6 to 10^8).  In my opinion, this implies the authors just swapped the words "TCID50" for "copies" without actually calculating their genomic standard curves properly.

Author Response

Please see the attachment

Author Response File: Author Response.pdf