Review Reports - CHO Cell-Produced Truncated Bovine Ephemeral Fever Virus Glycoprotein as a Promising Subunit Vaccine Candidate for Cattle

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

This revised manuscript has improved from the statistical analysis by including three cattle in the vaccination group and three in the sham group.

Two minor comments:

In the materials and methods and Results sections, please check all the statements describing the current data and make sure they stated the results are from three cattle in each group not two as in the previous manuscript. (eg. line 180-183, 189-190)
In Figure 4 and Figure 5, please label the y-axis and make sure they match the text and data in the Results.

Author Response

Dear Reviewer 1,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion have been addressed point by point.

In the materials and methods and Results sections, please check all the statements describing the current data and make sure they stated the results are from three cattle in each group not two as in the previous manuscript. (eg. line 180-183, 189-190)

Response: Thank you very much for pointing this out. The number of cattle has been revised throughout the manuscript.

In Figure 4 and Figure 5, please label the y-axis and make sure they match the text and data in the Results.

Response: Thank you for pointing this out. The Figure 4 and Figure 5 has been revised.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript investigates the immunogenicity of a subunit vaccine based on a truncated form of the BEFV G protein in cattle. The overall experimental design is rational, the conclusions are supported by experimental data, and the vaccine holds certain application prospects. However, the study still has several limitations that need further improvement and supplementation, with specific revision suggestions as follows:

The introduction section is somewhat verbose. Excessive descriptions are devoted to climate change and arbovirus transmission; it is recommended to streamline this part and highlight the hazards of BEFV and the research gaps in relevant vaccine development.
Details on the TCID₅₀assay method are not provided. The incubation conditions of serially diluted serum with the virus and the criteria for judging cytopathic effect (CPE) are not elaborated. It is recommended to clarify the CPE evaluation criteria (e.g., lesion degree, judgment time) in the neutralization test to improve the reproducibility of the experiment.
Methods for identifying the recombinant protein (e.g., Western blot, mass spectrometry) are not provided. Verifying the molecular weight merely by SDS-PAGE is insufficient to confirm the authenticity of the target protein. It is recommended to supplement the results of Western blot identification (using BEFV G protein-specific antibodies) and mass spectrometry for the recombinant protein to validate the identity and correctness of the target protein.
Add annotations for the names or implications of the vertical axes in Figure 6 and Figure 7.
The vaccine was administered three times on Day 0, Day 14 and Day 28, and neutralizing antibody titers were detected weekly over the 49-day experimental period, which is a rational design. Nevertheless, the IFN-γ levels were only measured up to Day 28 in Section 3.2.2; it is recommended to supplement the IFN-γ detection data after the third vaccination (Day 28).
The discussion section needs to be further in-depth, as it is currently rather descriptive. It is recommended to add discussions on the following issues:

6.1 Why is s510 more immunogenic than s521? Is it associated with glycosylation sites or structural stability?

6.2 Does the elevation of IFN-γ truly indicate a cytotoxic T lymphocyte (CTL) response? Are there any other indicators to support this finding?

6.3 What are the advantages and disadvantages of this study compared with existing research?

6.4 The lack of a viral challenge experiment fails to verify the protective efficacy of the vaccine. This study only detected neutralizing antibody titers and cytokine expression, but did not conduct a BEFV live virus challenge experiment, making it impossible to directly prove the actual protective effect of the s510 subunit vaccine in cattle. Inferring protective efficacy solely based on a neutralizing antibody titer of 1:32 lacks direct experimental evidence. If a supplementary challenge control experiment cannot be performed, it is recommended to clearly state this limitation in the discussion and propose that the core direction of subsequent research is to verify the protective efficacy via viral challenge.

Author Response

Dear Reviewer 2,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion and question have been addressed point by point.

The introduction section is somewhat verbose. Excessive descriptions are devoted to climate change and arbovirus transmission; it is recommended to streamline this part and highlight the hazards of BEFV and the research gaps in relevant vaccine development.

Response: Thank you for your suggestion. The hazards of BEFV including clinical signs and economic loss have been added on lines 40-48. The research gaps in relevant vaccine development was discussed on lines 108-112. With the shortened interval between BEFV outbreaks in many areas, climate change may be a key factor affect the distribution, abundance, and activity periods of the insect vectors to spread BEF, which increases the importance of controlling BEF in the near future. This statement was added on lines 49-66.

Details on the TCID₅₀assay method are not provided. The incubation conditions of serially diluted serum with the virus and the criteria for judging cytopathic effect (CPE) are not elaborated. It is recommended to clarify the CPE evaluation criteria (e.g., lesion degree, judgment time) in the neutralization test to improve the reproducibility of the experiment.

Response: Thank you for pointing this out. The details of the TCID₅₀ assay have been added to the Materials and Methods section on lines 222–227.

Methods for identifying the recombinant protein (e.g., Western blot, mass spectrometry) are not provided. Verifying the molecular weight merely by SDS-PAGE is insufficient to confirm the authenticity of the target protein. It is recommended to supplement the results of Western blot identification (using BEFV G protein-specific antibodies) and mass spectrometry for the recombinant protein to validate the identity and correctness of the target protein.

Response: Thank you for pointing this out. The process of performing the Western blot assay has been added to the Materials and Methods section on lines 176–194.

Add annotations for the names or implications of the vertical axes in Figure 4 and Figure 5.

Response: Thank you for pointing this out. The Figure 4 and Figure 5 has been revised.

The vaccine was administered three times on Day 0, Day 14 and Day 28, and neutralizing antibody titers were detected weekly over the 49-day experimental period, which is a rational design. Nevertheless, the IFN-γ levels were only measured up to Day 28 in Section 3.2.2; it is recommended to supplement the IFN-γ detection data after the third vaccination (Day 28).

Response: Thank you for pointing this out. IFN-γ regulates the Th1/Th2 balance to favor cell-mediated immunity and activates the pro-inflammatory function of macrophages. IFN-γ serves as a main regulator in the early stage of host immune system activation. Upregulation of IFN-γ was observed on Day 7 and Day 21, one week after vaccination. The early upregulation following vaccination indicates the antigenicity of this novel protein, which meets our expectations; therefore, longer follow-up of IFN-γ expression was considered unnecessary in this study.

The discussion section needs to be further in-depth, as it is currently rather descriptive. It is recommended to add discussions on the following issues:

Response: Thank you very much for providing these valuable suggestions. The responses to each suggestion and question have been addressed point by point in Discussion and Conclusion sections.

6.1 Why is s510 more immunogenic than s521? Is it associated with glycosylation sites or structural stability?

Response: Regarding Suggestion 6.1, the s510 protein was designed to increase protein yield in this study rather than to improve immunogenicity. We aim to preserve the antigenicity of s510 with high yield in this study. This information was added in lines 399-400 to prevent misunderstanding.

6.2 Does the elevation of IFN-γ truly indicate a cytotoxic T lymphocyte (CTL) response? Are there any other indicators to support this finding?

Response: Regarding Suggestion 6.2, using flow cytometry analysis is an effective way to identify the specific cells involved in this immune response. This information was added in lines 442-445 in Discussion section.

6.3 What are the advantages and disadvantages of this study compared with existing research?

Response: Regarding Suggestion 6.3, the advantages and disadvantages of this study were added to the Conclusion section in lines 460–465.

Response: The objective of this study is to evaluate the antigenicity of a novel protein s510. The s510 shows good results in neutralizing antibody elevation and cytokine upregulation, but it still requires further work to ensure its stability and to identify an appropriate adjuvant before proceeding to a commercial vaccine. This is the main reason a challenge test was not conducted in this study. This statement was added on lines 392-396 in the Discussion section.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript presents a timely and relevant study on the development of a subunit vaccine against Bovine Ephemeral Fever (BEF) using a truncated G protein expressed in a mammalian cell system. The authors address a significant gap in the field by evaluating the immunogenicity of a recombinant BEFV G protein in cattle, a species directly relevant to disease impact. The study is well-structured, and the data support the potential of the s510 antigen as a vaccine candidate. However, several issues need to be addressed to improve clarity, rigor, and impact before the manuscript can be considered for publication.

The study includes only three animals per group (vaccinated and sham). While the authors acknowledge this limitation in the Discussion, the small sample size significantly reduces the statistical power and generalizability of the findings. The authors should provide a rationale for the sample size or include a power analysis. Additionally, the use of non-parametric tests (Wilcoxon Two-Sample Test) is appropriate given the small n, but the results should be interpreted with caution.
In Section 2.4 (Animal Use and Ethics), the authors state that three cattle were included in the vaccination group and three in the sham group. However, the Results section (3.2.1 and 3.2.2) mentions only two cattle in the vaccinated group showing responses (Cattle A and B). This discrepancy needs clarification. Was one animal excluded? If so, why?
The study evaluates immunogenicity through neutralizing antibody titers and cytokine expression but does not include a viral challenge to assess protection. While the authors cite previous studies correlating antibody titers with protection, a challenge experiment would substantially strengthen the conclusion that s510 confers protective immunity. This should be addressed in the Discussion as a limitation and future direction.
The IFN-γ upregulation is presented as a key finding, but the data appear to be driven primarily by one animal (Cattle A). The authors should clarify whether the statistical significance remains when analyzing all three animals individually. If the response is not uniform, this should be discussed in the context of individual variability in vaccine response.

Author Response

Dear Reviewer 3,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion and question have been addressed point by point.

The study includes only three animals per group (vaccinated and sham). While the authors acknowledge this limitation in the Discussion, the small sample size significantly reduces the statistical power and generalizability of the findings. The authors should provide a rationale for the sample size or include a power analysis. Additionally, the use of non-parametric tests (Wilcoxon Two-Sample Test) is appropriate given the small n, but the results should be interpreted with caution.

Response: Thank you for pointing this out. The major limitation of this study is the low number of animals per group, as you mentioned. Because BEF is transmitted by biting midges and mosquitoes, this experiment needed to be conducted indoors to prevent natural infection. In Taiwan, most cows receive regular BEF inactivated vaccines when they are pregnant, so the maternal neutralizing antibody for BEF in calves remains at a high level and does not begin to decrease until four months of age, which increases the difficulty of this experiment. All experimental cattle in this study were around one year old, making them large in body size, with each weighing more than 300 kilograms. It is difficult to find a space to keep all six animals together indoors with insect-blocking equipment under current experimental regulations. The main contribution of this study is that it addresses the gap in evaluating the efficacy of recombinant BEF G protein-based subunit vaccines in cattle, which is difficult to address due to the low productivity of recombinant G protein produced by cell culture and the challenges associated with obtaining cattle under experimental conditions in previous studies.

In Section 2.4 (Animal Use and Ethics), the authors state that three cattle were included in the vaccination group and three in the sham group. However, the Results section (3.2.1 and 3.2.2) mentions only two cattle in the vaccinated group showing responses (Cattle A and B). This discrepancy needs clarification. Was one animal excluded? If so, why?

Response: We apologize for not correcting this in the previous manuscript. In this final version, there are three cattle in each group, and no cattle were excluded. The number of animals has been revised throughout the manuscript. We apologize for any misunderstanding.

The study evaluates immunogenicity through neutralizing antibody titers and cytokine expression but does not include a viral challenge to assess protection. While the authors cite previous studies correlating antibody titers with protection, a challenge experiment would substantially strengthen the conclusion that s510 confers protective immunity. This should be addressed in the Discussion as a limitation and future direction.

The IFN-γ upregulation is presented as a key finding, but the data appear to be driven primarily by one animal (Cattle A). The authors should clarify whether the statistical significance remains when analyzing all three animals individually. If the response is not uniform, this should be discussed in the context of individual variability in vaccine response.

Response: Thank you for pointing this out. Upregulation of IFN-γ was observed in all vaccinated cattle on Day 7 and in two of three vaccinated cattle on Day 21, indicating a consistent immune response in this study. Moreover, cattle A exhibited a stronger immune response than the other animals; swelling at the injection site measured 2 x 3 cm after the first vaccination, and greater upregulation of IFN-γ was observed in this animal, demonstrating individual variability following vaccination in this study. This information was added on lines 445–448 in the Discussion section.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have responded to all comments and made appropriate revisions to the manuscript.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The quality of the revised manuscript has significantly improved.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor

Vaccines

On reviewing the article vaccines-3905389, it could be concluded that the article might be publishable after making few corrections:

- Extensive language editing should be done. The entire manuscript must be written in past passive tense EXCEPT when facts are mentioned (in discussion only).

- Avoid redundancy e.g. lines 40-42 are typically repeated in abstract. Please consider throughout.

- Please expand the role of vectors in sections of introduction and discussion.

- References are few. Please add more recent citations particularly in introduction, methodology and discussion.

Comments on the Quality of English Language

- Extensive language editing should be done. The entire manuscript must be written in past passive tense EXCEPT when facts are mentioned (in discussion only).

- Avoid redundancy e.g. lines 40-42 are typically repeated in abstract. Please consider throughout.

Author Response

Dear Reviewer 1,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion and question have been addressed point by point.

- Extensive language editing should be done. The entire manuscript must be written in past passive tense EXCEPT when facts are mentioned (in discussion only).

Response: Thank you for point this out. The article has been revised following your suggestion.

- Avoid redundancy e.g. lines 40-42 are typically repeated in abstract. Please consider throughout.

Response: Thank you for point this out. The redundancy has been revised.

- Please expand the role of vectors in sections of introduction and discussion.

Response: Thank you for the valuable insights on this article. The role of the vector was expanded upon in the discussion section. (Line 361-364). The reason for choosing a mammalian cell expression system to produce this vaccine has been added to the introduction section. (Line 89-104).

- References are few. Please add more recent citations particularly in introduction, methodology and discussion.

Response: Thank you for point this out. The article has been revised and added more references following your suggestion.

Comments on the Quality of English Language

- Extensive language editing should be done. The entire manuscript must be written in past passive tense EXCEPT when facts are mentioned (in discussion only).

Response: Thank you for point this out. The article has been revised following your suggestion.

- Avoid redundancy e.g. lines 40-42 are typically repeated in abstract. Please consider throughout.

Response: Thank you for point this out. The redundancy has been revised.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript entitled “CHO Cell–Produced Truncated Bovine Ephemeral Fever Virus Glycoprotein as a Promising Subunit Vaccine Candidate for Cattle”, authors attempt to generate BEF glycoprotein-based subunit vaccine and described their primary findings on the immunogenicity of the truncated proteins designated s510 and s521. And they showed that “the recombinant protein s510 is a promising antigen for future BEF subunit vaccine development.” The manuscript is well written; Nevertheless, I found that the experiments were not carefully designed, data were not well-organized, some critical results are missing. The scientific merits in the manuscript are scarce.

According to introduction section, Line 69-70. There are inactivated vaccines for BEF are currently available on the market. Authors should include at least one of the inactivated vaccines as a sham group, compare the antibody titer and cytokine levels of them, which would help to demonstrate the superior of the vaccine designed over the inactivated vaccine.
Cytokine examination, Line 221-222. Author claimed that “IL-4, IL-10, IL-12B, IFN-γ, TNF-α, and β-actin were included for cytokine detection “, however, I only noticed the description IFN-γ level (Line 321-337). Results of the rest four cytokines are missing in the text. Further, The IFN-γ mRNA levels in blood are less reliable than those measured by the ELISpot assay, which quantifies IFN-γ secreting lymphocytes in peripheral blood mononuclear cells (PBMCs).
In the “Animal use and Ethics” section. Line 189-192. Author describes how experimental groups were conducted. It would be better to included it in the vaccine scheme section.
The Materials and Methods section is excessively long and includes unnecessary details. Please provide a concise description of the key operational steps and methodologies, focusing on the CHO cell expression component: (1) whether transient or stable transfected cell was used, (2) whether bulk pool or mini pool CHO cells were employed, and (3) whether a temperature shift and/or fed-batch was implemented during the 14-day culture process.
After 35 days of immunization with the subunit vaccine, the neutralizing antibody titer can reach 16 to 32-fold. Whether this antibody provides robust immune-protection? In the case of BEF, what level of antibody titer is considered to provide immune protection? Given the relatively short duration of antibody persistence induced by subunit vaccines, this study should determine how long the protective antibody titer can be maintained.
Figure 4. The authors did not specify the number of animals used per group. Were only two animals used in both Cattle A and Cattle B groups? If only two animals were used per group, the results would lack statistical significance. If more than three animals were used, standard deviation should be indicated on the bar graph.
What accounts for the significant difference in antibody levels between the Vaccinated Cattle A and Vaccinated Cattle B groups at 35 days? Please discuss it in the discussion section.
The Methods section describes the processing of both CHO cell supernatant and pellet (P149-P155). However, the Results section only presents the SDS-PAGE analysis of the target protein from the supernatant. Please supplement with the corresponding electrophoretic data for the pellet.
Vaccination groups, Line 181-182. “To ensure the subunit vaccine without any safety issue, local and systemic hypersensitive reaction was monitored by veterinarian every day”. This should be clearly specified and presented using clinical scoring system.

Author Response

Dear Reviewer 2,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion and question have been addressed point by point.

According to introduction section, Line 69-70. There are inactivated vaccines for BEF are currently available on the market. Authors should include at least one of the inactivated vaccines as a sham group, compare the antibody titer and cytokine levels of them, which would help to demonstrate the superior of the vaccine designed over the inactivated vaccine.

Response: Thank you for the suggestion. In this study, we focus on evaluating the antigenicity of a novel antigen, S510. While we acknowledge that the inactivated BEF vaccine is effective, incorporating it into our current study would introduce uncontrollable variables, such as different adjuvants, other structural proteins, or unlabeled ingredients present in the inactivated vaccine. Our primary objective is to assess the potential of this novel antigen as a subunit vaccine candidate, rather than comparing the efficacy of this experimental subunit vaccine to that of the commercial inactivated vaccine.

Cytokine examination, Line 221-222. Author claimed that “IL-4, IL-10, IL-12B, IFN-γ, TNF-α, and β-actin were included for cytokine detection “, however, I only noticed the description IFN-γ level (Line 321-337). Results of the rest four cytokines are missing in the text. Further, The IFN-γ mRNA levels in blood are less reliable than those measured by the ELISpot assay, which quantifies IFN-γ secreting lymphocytes in peripheral blood mononuclear cells (PBMCs).

Response: Thank you for point this out. The expression levels of the cytokines IL-4, IL-10, IL-12B, and TNF-α did not show any significant differences in this study. And this sentence was added in result section on Line 322-323. For the reliability of using IFN-γ mRNA levels presented the real IFN-γ protein produced in PBMCs, there is a research indicated a high correlation between IFN-γ mRNA gene in SYBR Green-based qPCR and IFN-γ protein ELIspot quantification in human PBMCs in 2022, this reference has been added in Discussion part (Line 405-410).

In the “Animal use and Ethics” section. Line 189-192. Author describes how experimental groups were conducted. It would be better to included it in the vaccine scheme section.

Response: Thank you for the suggestion. This part has been moved to Vaccine Scheme section.

The Materials and Methods section is excessively long and includes unnecessary details. Please provide a concise description of the key operational steps and methodologies, focusing on the CHO cell expression component: (1) whether transient or stable transfected cell was used, (2) whether bulk pool or mini pool CHO cells were employed, and (3) whether a temperature shift and/or fed-batch was implemented during the 14-day culture process.

Response: Thank you for your suggestion. The Materials and Methods section has been revised and condensed from five pages to 3.5 pages. It has been reorganized into the following subsections: 2.1 Vector Construct and Protein Expression, 2.2 Protein Purification and Quantification, 2.3 Vaccine Scheme, 2.4 Animal Use and Ethics, 2.5 Neutralization Test, 2.6 Cytokine Examination, 2.7 BEFV Detection, and 2.8 Statistical Analysis.

Regarding protein expression details, these four proteins were expressed in transiently transfected cells using a bulk pool of ExpiCHO-S cells. The culture temperature was shifted from 37°C to 32°C on Day 3. No bovine serum was added; instead, ExpiCHO Feed and ExpiFectamine CHO Enhancer were used according to the manufacturer's instructions of ExpiCHO expression system in this study.

After 35 days of immunization with the subunit vaccine, the neutralizing antibody titer can reach 16 to 32-fold. Whether this antibody provides robust immune-protection? In the case of BEF, what level of antibody titer is considered to provide immune protection? Given the relatively short duration of antibody persistence induced by subunit vaccines, this study should determine how long the protective antibody titer can be maintained.

Response: There is no established gold standard for the antibody titer level considered sufficient to provide immune protection against BEF infection. Protective antibody titers ranging from 1:4 to 1:64 have been regarded as effective for preventing BEF infection. Based on a large-scale retrospective study of BEF occurrence and neutralizing antibody titers in cattle populations from 2001 to 2014 in Taiwan, neutralizing antibody titers below 1:32 were associated with clinical disease during BEF outbreaks; according to studies conducted there, a neutralizing antibody titer higher than 1:32 is considered protective against BEF infection in cattle. The description was added in discussion section on Line 379-388. In this study, the neutralizing antibody titer reached 1:32 by Day 35 and persisted at least until Day 49 during the experimental period. The primary objective of this study is to evaluate the capacity of the novel antigen s510 as a subunit vaccine candidate; the duration of the protective immune response is a separate consideration from the objective of this study.

Figure 4. The authors did not specify the number of animals used per group. Were only two animals used in both Cattle A and Cattle B groups? If only two animals were used per group, the results would lack statistical significance. If more than three animals were used, standard deviation should be indicated on the bar graph.

Response: There were only two cattle in the vaccinated group. The same two animals were also included in the sham groups. Both the sham and experimental treatments were conducted on the same cattle, on the same date but in different years, to minimize potential variations due to differences in the animals and seasonal factors. The antigenicity of s510 was assessed in these two cattle, both exhibiting 16- to 32-fold increases in neutralizing antibody titers, indicating promising antigenic potential for this novel protein. To refine the experiment and minimize the number of animals used in accordance with the 3R principles, only these two cattle were included in the study. The results also highlight individual differences in immune response following vaccination. We believe that using two cattle is sufficient to demonstrate the capacity of the new antigen s510 as a promising subunit vaccine candidate in this study. While including more animals would provide stronger statistical evidence, statistical analysis is not the primary focus of this research. The rising neutralizing antibody levels and upregulation of IFN-γ in all vaccinated cattle demonstrate promising results for this vaccine.

What accounts for the significant difference in antibody levels between the Vaccinated Cattle A and Vaccinated Cattle B groups at 35 days? Please discuss it in the discussion section.

Response: Significant differences in neutralizing antibody titers were observed between the Vaccinated Group and the Sham Group. Vaccinated Cattle A and Vaccinated Cattle B both belong to the Vaccinated Group; therefore, comparisons were made between groups, not within the same group. However, we noted a variant immune response in Vaccinated Cattle A, characterized by slight swelling at the injection site, high IFN-γ expression, and low neutralizing antibody titer. This observation has been added to the discussion section (Lines 412-416).

The Methods section describes the processing of both CHO cell supernatant and pellet (P149-P155). However, the Results section only presents the SDS-PAGE analysis of the target protein from the supernatant. Please supplement with the corresponding electrophoretic data for the pellet.

Response: This study focuses on soluble proteins as subunit vaccine candidates. Therefore, we collected only the supernatants, not the pellets. The description of P149-155 referred to the location of recombinant proteins in their original form (within cells) and their secreted form (in the medium). To clarify the article, this content has been deleted and revised in Vector Construct and Protein Expression section.

Vaccination groups, Line 181-182. “To ensure the subunit vaccine without any safety issue, local and systemic hypersensitive reaction was monitored by veterinarian every day”. This should be clearly specified and presented using clinical scoring system.

Response: Thank you for point this out. The sentence has been revised to “To ensure the subunit vaccine without any safety issue, local (redness, swelling, heat, or pain at the injection site) and systemic hypersensitive reaction (fever) was monitored by veterinarian every day.” (Line 191-193)

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, Hsu and colleagues present a study on the development of bovine ephemeral fever (BEF) G protein subunit vaccines and their antigenicity. The vaccination study with the candidate protein s510 was conducted on two cattle, and antigenicity was measured by neutralization assay and cytokine IFN-γ determination. The recombinant protein construct presented in this manuscript demonstrates high yield from the CHO cell expression system with elicitation of neutralizing antibodies. However, a major limitation is that the low number of animals and the repeat use of the same two cattle impair the statistical power in the data interpretation.

Comments:

Is there a specific reason that only two cattle were used for both the control and vaccinated groups? As the authors mentioned in the introduction, early studies from references 14 and 15 investigated G protein-based BEF vaccines using cohorts of at least 6 and 10 cattle, respectively. Using a cohort of only two cattle is insufficient to generate adequate statistical power to support the antigenicity results.
Continuing from point 1, given that only two subunit vaccines have been studied since last century, it would be clearer and more informative to provide a comparison and discussion of the advantages of the current vaccine design relative to these historical studies.
The conventional abbreviation for kilodalton is kDa.
In Figure 3, it is unclear how the levels of s510 and s521 were determined. The bands may not fall within the linear range of the BSA standard curve, making extrapolation needs to be cautious. Additionally, it is also not explained how the protein levels shown on the gel could be calculated to reflect the final yield of 30 mg/L. Please add sufficient description and explanation.
In Figure 4 and 5, please specify what statistical testing was used. Given that the same two cattle were used in both control and vaccinated groups, was the p-value determined from the paired test?

Author Response

Dear Reviewer 3,

Thank you for reviewing this paper and providing valuable feedback.

The responses to each suggestion and question have been addressed point by point.

Comments:

Is there a specific reason that only two cattle were used for both the control and vaccinated groups? As the authors mentioned in the introduction, early studies from references 14 and 15 investigated G protein-based BEF vaccines using cohorts of at least 6 and 10 cattle, respectively. Using a cohort of only two cattle is insufficient to generate adequate statistical power to support the antigenicity results.

Response: Yes. The antigenicity of s510 was evaluated in two cattle, both of which exhibited 16- to 32-fold increases in neutralizing antibody titers, indicating promising antigenic potential for this novel protein. To refine the experiment and minimize the number of animals used in accordance with the 3R principles (Replacement, Reduction, and Refinement), only these two cattle were included in the study. In references 14 and 15, six to ten cattle were used to demonstrate the antigenicity of the original virus G protein, which was derived either from the virus virion or a recombinant vaccinia virus. The antigenicity of the truncated G protein of BEFV produced by a mammalian cell expression system has been demonstrated in mice and guinea pigs in other studies. The present study evaluates the antigenicity of a truncated recombinant protein expressed in mammalian cells in cattle, which is the primary distinction from previous work. We believe that using two cattle is sufficient to demonstrate the capacity of the new antigen s510 as a promising subunit vaccine candidate in this study. While including more animals would provide stronger statistical evidence, statistical analysis is not the primary focus of this research. The rising neutralizing antibody levels and upregulation of IFN-γ in all vaccinated cattle demonstrate promising results for this vaccine.

Continuing from point 1, given that only two subunit vaccines have been studied since last century, it would be clearer and more informative to provide a comparison and discussion of the advantages of the current vaccine design relative to these historical studies.

Response: Thank you for the valuable insights on this article. The advantages and challenges of current vaccine research have been added to the introduction section (Line 89-104).

The conventional abbreviation for kilodalton is kDa.

Response: Thank you for this reminder. The abbreviation for kilodalton has been changed to kDa throughout the article.

In Figure 3, it is unclear how the levels of s510 and s521 were determined. The bands may not fall within the linear range of the BSA standard curve, making extrapolation needs to be cautious. Additionally, it is also not explained how the protein levels shown on the gel could be calculated to reflect the final yield of 30 mg/L. Please add sufficient description and explanation.

Response: We understand that there are minor errors associated with the true value in extrapolation analysis. The primary objective of the concentration determinant in this study is to identify the recombinant protein with the highest productivity among four candidates, rather than to achieve absolute quantification for manufacturing improvement or other purposes. Therefore, we believe that extrapolation analysis is sufficient for this study.

The following content has been added to the Materials and Methods section, lines 170–176, to describe the concentration determinant. “The concentration of recombinant proteins was determined using extrapolation test with standard bovine serum albumin (BSA) in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The peak volume of the standard BSA was identified and the volume of the target proteins also measured at 70 kilo Dalton (kDa), which corresponds to the expected size of the recombinant antigen. After linearizing the BSA volumes, the expression level of the target proteins was deter-mined.”

In Figure 4 and 5, please specify what statistical testing was used. Given that the same two cattle were used in both control and vaccinated groups, was the p-value determined from the paired test?

Response: Thank you for point this out. The statistical analysis was added in figure 4 and 5. The p-value is determined from the paired test

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Main concerns are explained or revised. The updated manuscript is now acceptable.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed my questions in the revised version.