Modern Strategies for Brucellosis Vaccination: From Traditional Approaches to Innovative Platforms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I have read with interest the submitted review which systematically analyze modern strategies for brucellosis vaccination in livestock and humans, with emphasis on comparative evaluation of the efficacy and safety of existing vaccine platforms, and examines live attenuated, inactivated, subunit, vector, and DNA vaccines, their immunological mechanisms of action, advantages, and limitations of application. However, there are major specific points that should be addressed to enhance the quality and precision of the work.

Major Comments:

1. The search was limited to PubMed, Scopus, and Web of Science. Other databases (e.g., regional or specialized veterinary databases) were not included, potentially missing relevant studies.Although reports from WHO and World Organisation for Animal Health were used, other grey literature (e.g., theses, government reports, non-indexed data) may not be fully covered.
2. While the review covers both veterinary and human medicine, human brucellosis vaccine data remain scarce. Most findings are likely based on animal studies, limiting translation to humans.
3. The strong link between animal infection and human disease means: controlling brucellosis requires integrated “One Health” approaches.However, coordination between sectors (veterinary, medical, environmental) is often limited.and there is no approved vaccine for humans, which is a major gap in brucellosis control.
4. From over 300 studies, only the “most significant and cited” were included. This subjective filtering may: Omit relevant but less-cited studies  and Favor well-known research over newer or niche findings.
5. Inclusion criteria emphasize animal and preclinical studies, with relatively fewer human trials. This limits the applicability of conclusions to human vaccination strategies.
6. The methodology describes a systematic search, but not a meta-analysis. This means: No pooled statistical estimates and conclusions are largely qualitative.
7. Individual Brucella antigens (e.g., Omp16, Omp19, L7/L12, Cu/Zn-SOD) are mmunogenic, but do not provide sufficient protection alone, this necessitates complex multicomponent vaccines, increasing development difficulty.

Minor comments:

8. Line 26: The review examines live attenuated, inactivated, subunit, vector, and DNA vaccines, their immunological mechanisms of action, advantages, and limitations of application. Corrected: The review examines live attenuated, inactivated, subunit, vector, and DNA vaccines, as well as their immunological mechanisms of action, advantages, and limitations of application.

9. Line 27: ‘This information allows better understanding of protective immunity formation mechanisms’ Corrected ‘This information allows a better understanding of the mechanisms of protective immunity formation’

9. Line 77: “sanitary-hygienic practices” → correct but could be simplified to “sanitation and hygiene practices” for readability.
10. Line 97: “databases PubMed, Scopus, and Web of Science” ✔ Correction: “databases such asPubMed, Scopus, and Web of Science”
11. Line 102: “used for literature search” ✔ Correction: “used for theliterature search”
12. Line 103-104: “DNA vaccine Brucella, subunit vaccine Brucella, DIVA vaccine brucellosis” ✔ Correction: “Brucella DNA vaccine, Brucella subunit vaccine, DIVA vaccine for brucellosis”
13. Line 111: “At the first stage, more than 300 publications were identified, of which after assessing relevance and study quality, the most significant and cited works were included in the review”  its is unclear structure and missing commas, Correction: “At the first stage, more than 300 publications were identified. After assessing relevance and study quality, the most significant and highly cited works were included in the review.”

14. Line 124: “limited capabilities for epizootic control” Corrected: “limited capacity for epizootic control”
15. Live attenuated vaccines are the most effective method… Live attenuated vaccines are the most effective vaccines…” Combine: “Live attenuated vaccines are the most effective method for controlling animal brucellosis [27].” Avoid repeating the same phrase

16. Line 200: “three out of five goats were protected from infection” More formal: “three of five goats were protected from infection”

Author Response

Comments 1: The search was limited to PubMed, Scopus, and Web of Science. Other databases (e.g., regional or specialized veterinary databases) were not included, potentially missing relevant studies.Although reports from WHO and World Organisation for Animal Health were used, other grey literature (e.g., theses, government reports, non-indexed data) may not be fully covered.

Response 1: We sincerely thank the Reviewer for this important and constructive comment regarding our literature search strategy. We fully agree that restricting the search to PubMed, Scopus, and Web of Science, along with a focus on English-language publications, may have excluded certain regional studies, dissertations, and national veterinary reports. We acknowledge this as a recognized methodological limitation of the present review.

The selection of these databases was guided by their widespread recognition in international scientific practice as repositories of rigorously peer-reviewed research with transparent methodologies. This approach aligns with established standards for conducting high-quality systematic and narrative reviews at an international level. To supplement our search and capture relevant grey literature, we additionally analyzed official reports from the World Health Organization (WHO) and the World Organisation for Animal Health (WOAH), and performed backward citation tracking of key publications.

Revisions made to the manuscript:

A dedicated subsection "8. Limitations of the Review" has been added (Section 8, page 12, lines 517-532) to transparently discuss the potential impact of excluding regional veterinary databases and unpublished materials on the comprehensiveness of the literature coverage.

To demonstrate our commitment to incorporating regionally relevant data and partially mitigate geographical bias, we have integrated verified epidemiological data from the Republic of Kazakhstan into Section 3 ("Prevalence of Brucellosis in Animals and Humans") (Section 3, page 4, lines 155-167). Brucellosis has remained endemic in Kazakhstan since the 1930s, with over 1,300 human cases reported annually (~7.6 per 100,000 population). Livestock seropositivity stands at approximately 0.6% in cattle and 0.4% in small ruminants [12]. Recent surveillance (2020–2024) identified a focal distribution: 1,314 out of 2,460,956 tested small ruminants reacted positively (0.05%), with up to 78.6% of cases concentrated in specific regions [13, 14]. Key risk factors include the consumption of unpasteurized dairy products and direct contact with aborted materials, exacerbated by the predominance of smallholder farming systems.

In Section 2 ("Literature Search Methodology") (page 3, lines 106-111), we have added a methodological note clarifying that future updates of this review will consider expanding the search strategy to include specialized veterinary databases (e.g., CAB Abstracts, AGRIS) and national repositories, contingent upon resources for critical appraisal and translation.

We believe these revisions significantly enhance the methodological transparency and practical relevance of the review for highly endemic regions. We sincerely thank the Reviewer for this valuable suggestion, which has substantially improved the quality and rigor of our manuscript。

 

Comments 2: While the review covers both veterinary and human medicine, human brucellosis vaccine data remain scarce. Most findings are likely based on animal studies, limiting translation to humans.

Response 2: We sincerely thank the Reviewer for this valuable observation. We fully agree that the distinction between veterinary/preclinical evidence and human translational potential required clearer framing throughout the manuscript. To address this transparently, we have implemented three targeted revisions:

In Section 5 ("Brucellosis Vaccine Development") (page 5, lines 196–203), immediately following the statement on the absence of licensed human vaccines, we added a clarifying paragraph emphasizing that most efficacy data derive from animal models, and that direct extrapolation to humans requires caution due to species-specific immunological differences. We explicitly state that formulations such as "translational potential," "data extrapolation," and "limited human evidence base" are used to distinguish clinical from preclinical findings.

In Section 8 ("Limitations of the Review") (page 12, lines 533–539), we added a dedicated subsection "Translational considerations" acknowledging that immunobiological differences (MHC haplotypes, cytokine profiles, intracellular Brucella trafficking) limit direct application of veterinary data to human contexts. We clarify that protective efficacy observed in livestock or rodent models should be interpreted as indicative rather than definitive for human vaccine development, and emphasize the need for dedicated preclinical studies in relevant humanized models before clinical translation.

In Section 7 ("Promising Directions and New Technologies...") (page 12, lines 502–508), following the "One Health" discussion, we inserted a paragraph on "Human-oriented vaccine platforms" highlighting subunit, DNA, and vector-based candidates currently undergoing preclinical evaluation. We note their advantages in safety and DIVA compatibility, while underscoring that clinical data in humans remain scarce and that continued investment under the One Health framework is essential.

These additions ensure transparent, balanced reporting of evidence levels and prevent overinterpretation of preclinical data for human applications. We believe this significantly strengthens the methodological rigor, translational clarity, and practical relevance of the review. We are grateful for this constructive suggestion

 

Comments 3: The strong link between animal infection and human disease means: controlling brucellosis requires integrated “One Health” approaches.However, coordination between sectors (veterinary, medical, environmental) is often limited.and there is no approved vaccine for humans, which is a major gap in brucellosis control.

Response 3: We sincerely thank the Reviewer for highlighting these critical systemic challenges. We fully agree that effective brucellosis control is impossible without integrated "One Health" strategies, and that the absence of a licensed human vaccine remains a major global health gap.

Following your recommendation, we have substantially strengthened and clarified these aspects in the revised manuscript:

One Health implementation barriers: In Section 7 ("Promising Directions and New Technologies..."), we have expanded the discussion on the "One Health" approach to explicitly address institutional, communicational, and resource barriers that limit cross-sectoral coordination between veterinary, medical, and environmental services (Section 7, page 11, lines 496-501).

Human vaccine gap: In the Conclusion, we now explicitly state that the lack of an approved human brucellosis vaccine represents a strategic challenge, driven by ethical constraints on clinical trials, safety concerns with live attenuated strains, and limited commercial investment. We emphasize that current human protection relies entirely on veterinary control, and highlight subunit, mRNA, and viral-vector platforms as the most promising candidates for future human use (Conclusion, page 13, lines 575-582).

Actionable recommendations: We have added specific proposals to strengthen interagency surveillance data sharing, harmonize animal vaccination protocols, and establish international consortia to accelerate translational research on human brucellosis vaccines.

These revisions ensure that the review more clearly addresses systemic challenges and provides a forward-looking perspective on bridging the human vaccine gap within a One Health framework. We are grateful for this valuable observation, which significantly improved the practical relevance of our manuscript. 

Comments 4: From over 300 studies, only the “most significant and cited” were included. This subjective filtering may: Omit relevant but less-cited studies  and Favor well-known research over newer or niche findings.

Response 4: We thank the Reviewer for this methodological comment. Study selection prioritized methodological quality, thematic relevance, and contribution to vaccine development insights—not citation count alone. Recent and regionally relevant studies were deliberately included despite lower visibility. We have clarified these criteria in Section 2 (page 3, lines 116-125) and acknowledged the narrative review's representative (rather than exhaustive) scope.

Comments 5: Inclusion criteria emphasize animal and preclinical studies, with relatively fewer human trials. This limits the applicability of conclusions to human vaccination strategies.

Response 5: We sincerely thank the Reviewer for this precise observation. We fully agree that the evidence base for brucellosis vaccines is predominantly derived from animal and preclinical studies, with very limited human trial data. This reflects the current reality of the field: no licensed human vaccine exists, and ethical/regulatory constraints restrict clinical trials. To address this transparently, we added a methodological note in Section 2 clarifying that our inclusion criteria mirror the existing evidence landscape rather than selection bias. We also inserted a “Translational considerations” paragraph in Section 7 acknowledging that reliance on animal data inherently limits direct applicability to human strategies, and refined the Conclusion to clearly distinguish veterinary-proven platforms from those with human translational potential (Section 2, page 4, lines 126–132; Conclusion, page 13, lines 556–564). These revisions enhance transparency and help readers accurately interpret the scope of our conclusions.

Comments 6: The methodology describes a systematic search, but not a meta-analysis. This means: No pooled statistical estimates and conclusions are largely qualitative.  

Response 6: We sincerely thank the Reviewer for this precise methodological observation. We fully agree that this review employs a systematic literature search but does not include a meta-analysis with pooled statistical estimates.

The heterogeneity of included studies—spanning different Brucella species, vaccine platforms, animal models, outcome measures, and study designs—precluded meaningful quantitative pooling. Such methodological diversity is characteristic of the brucellosis vaccine field and is better addressed through narrative synthesis that allows contextual interpretation of immunological mechanisms, safety profiles, and translational potential.

To address this transparently, we have added a dedicated methodological note to Section 2 ("Literature Search Methodology") (Section 2, page 4, lines 133-144), explicitly stating that this is a narrative review with systematic search elements, and that conclusions are based on qualitative synthesis rather than statistical meta-analysis. We also acknowledge that the absence of pooled estimates limits the ability to quantify effect sizes across vaccine platforms, but emphasize that the narrative approach enables comprehensive evaluation of mechanistic insights essential for guiding future vaccine development.

We believe this clarification enhances methodological transparency and helps readers appropriately interpret the scope and conclusions of the review. We are grateful for this constructive comment.

Comments 7: Individual Brucella antigens (e.g., Omp16, Omp19, L7/L12, Cu/Zn-SOD) are mmunogenic, but do not provide sufficient protection alone, this necessitates complex multicomponent vaccines, increasing development difficulty.

Response 7: We sincerely thank the Reviewer for this precise observation. We fully agree that while individual Brucella antigens demonstrate immunogenicity, monocomponent formulations generally fail to confer robust protection—necessitating multicomponent vaccine constructs.

To address this, we have added clarifying text to Section 6 explaining that the limited efficacy of single-antigen vaccines reflects Brucella's complex intracellular pathogenesis, requiring coordinated humoral and Th1 responses. We also added a paragraph to Section 7 highlighting how vector-based, mRNA, and nanoparticle platforms enable practical co-delivery of multiple antigens, mitigating traditional developmental complexity (Section 7, page 12, lines 509-516).

These revisions provide a clearer rationale for multicomponent strategies and help readers interpret translational challenges in brucellosis vaccine development.

Comments 8: Line 26: The review examines live attenuated, inactivated, subunit, vector, and DNA vaccines, their immunological mechanisms of action, advantages, and limitations of application. Corrected: The review examines live attenuated, inactivated, subunit, vector, and DNA vaccines, as well as their immunological mechanisms of action, advantages, and limitations of application. 

Response 8: The sentence has been revised for clarity and grammatical correctness. The suggested wording has been adopted.

The review examines live attenuated, inactivated, subunit, vector, and DNA vaccines, as well as their immunological mechanisms of action, advantages, and limitations of application (Section Abstract, page 1, lines 25-26).

Comments 9. Line 27: ‘This information allows better understanding of protective immunity formation mechanisms’ Corrected ‘This information allows a better understanding of the mechanisms of protective immunity formation.

Response 9. The sentence has been corrected to: “This information allows a better understanding of the mechanisms of protective immunity formation.” (Section Abstract, page 1, lines 27-28).

Comments 9. Line 77: “sanitary-hygienic practices” → correct but could be simplified to “sanitation and hygiene practices” for readability. Response 9. The term has been revised to “sanitation and hygiene practices” to improve readability. (Section 1, page 3, lines 77).

Comments 10. Line 97: “databases PubMed, Scopus, and Web of Science” ✔ Correction: “databases such as PubMed, Scopus, and Web of Science.

Response 10. The sentence has been corrected to: “databases such as PubMed, Scopus, and Web of Science.” (Section 1, page 3, lines 96-97).

Comments 11. Line 102: “used for literature search” ✔ Correction: “used for the literature search”

Response 11. The phrase has been corrected to: “used for the literature search.” (Section 1, page 3, lines 102).

Comments 12. Line 103-104: “DNA vaccine Brucella, subunit vaccine Brucella, DIVA vaccine brucellosis” Correction: “Brucella DNA vaccine, Brucella subunit vaccine, DIVA vaccine for brucellosis”

Response 12. The keywords have been revised to: “Brucella DNA vaccine, Brucella subunit vaccine, DIVA vaccine for brucellosis.” (Section 1, page 3, lines 103-104)

Comments 13. Line 111: “At the first stage, more than 300 publications were identified, of which after assessing relevance and study quality, the most significant and cited works were included in the review”  its is unclear structure and missing commas, Correction: “At the first stage, more than 300 publications were identified. After assessing relevance and study quality, the most significant and highly cited works were included in the review.”

Response 13. The sentence has been restructured for clarity: “At the first stage, more than 300 publications were identified. After assessing relevance and study quality, the most significant and highly cited works were included in the review.” (Section 2, page 3, lines 111).

Comments 14. Line 124: “limited capabilities for epizootic control” Corrected: “limited capacity for epizootic control” 

Response 14. The term has been corrected to “limited capacity for epizootic control.” (Section 2, page 4, lines 124)

Comments 15. Live attenuated vaccines are the most effective method… Live attenuated vaccines are the most effective vaccines…” Combine: “Live attenuated vaccines are the most effective method for controlling animal brucellosis [27].” Avoid repeating the same phrase

Response 15. The sentence has been revised to avoid repetition:
“Live attenuated vaccines are the most effective method for controlling animal brucellosis [31].” (Section 5.1, page 5, lines 210)

Comments 16. Line 200: “three out of five goats were protected from infection” More formal: “three of five goats were protected from infection”.   

Response 16. The sentence has been revised to: “three of five goats were protected from infection.” (Section 5, page 5, lines 200)

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive review of modern strategies for brucellosis vaccination, covering classical and emerging platforms, including live attenuated, inactivated, subunit, vector, and DNA vaccines. The topic is highly relevant given the continued global burden of brucellosis and the lack of an approved human vaccine. The integration of the “One Health” perspective is particularly valuable.

Overall, the manuscript is informative and timely; however, major revisions are required to improve methodological rigor, critical analysis, and clarity. The current version reads more as a narrative overview rather than a systematic or critical review, despite claims of systematic methodology.

In this way, my recommendation is, either restructure the manuscript as a narrative review, or strengthen methodology by adding a PRISMA flow diagram, providing detailed search strings including study selection criteria and screening process.

Additional comments:

• The manuscript largely summarizes existing literature without sufficient critical evaluation. Furthermore, the section on influenza vector vaccines (Flu-BA) is disproportionately detailed and presented very positively. Major concerns are about potential institutional bias (authors affiliated with developing institute), and it should be clearly acknowledge potential conflict of interest in interpretation. Authors should add limitations of Flu-BA platform, independent validation studies (if available), and comparison with other vector platforms.
• Referencing issues: some references appear outdated or not critically contextualized, also correct inconsistent formatting (e.g., capitalization, author names).

Author Response

Comments 1: In this way, my recommendation is, either restructure the manuscript as a narrative review, or strengthen methodology by adding a PRISMA flow diagram, providing detailed search strings including study selection criteria and screening process.

Response 1:

We appreciate the Reviewer’s vigilance regarding critical balance and transparency. We acknowledge that the initial presentation of the Flu-BA platform lacked sufficient critical context. To address this, we have implemented the following revisions:

1. Added a dedicated subsection in Section 5.4 titled “Critical evaluation and comparative context of vector platforms”, which explicitly outlines limitations of the Flu-BA system, including potential pre-existing/vector-induced immunity, challenges in scalable manufacturing and process standardization, evolving regulatory frameworks, and the need for independent multi-center validation (Sections 5.4, page 8, lines 336-353).
2. Expanded the discussion to include a comparative analysis of Flu-BA versus alternative viral vector platforms (adenovirus, Salmonella, poxvirus systems), highlighting distinct trade-offs in cargo capacity, cellular tropism, and manufacturing scalability [71] (page 18, lines 789-790).
3. Added Section 5.6 (Limitations of Current Vaccine Platforms) providing a balanced, platform-by-platform critical synthesis of safety, immunogenicity, and translational constraints (Sections 5.6, page 9, lines 379-404).  .
4. Strengthened the Conflicts of Interest section with a clarifying statement affirming that all claims regarding Flu-BA are grounded in peer-reviewed, publicly available data, and we explicitly welcome independent validation studies. These changes ensure a more objective, critically balanced presentation of all vaccine platforms discussed (Conflicts of Interest, page 14, lines 604–608).

Comments 2: Referencing issues: some references appear outdated or not critically contextualized, also correct inconsistent formatting (e.g., capitalization, author names).

Response 2: We sincerely thank the reviewer for this insightful comment. In response, we have thoroughly revised the relevant sections to ensure critical contextualization and to integrate recent literature (2020–2025). Foundational studies from the 1990s are now explicitly framed as historical benchmarks, with their limitations carefully contextualized in light of contemporary systematic reviews, meta-analyses, and research on next-generation delivery platforms [57, 62, 64–66, 69]. We have also verified that all in-text citations explicitly specify the study design, model system, or translational relevance where appropriate. The complete reference list has been updated to strictly comply with the Vaccines (MDPI) formatting guidelines, including consistent capitalization style, standardized author name formatting, correct journal abbreviations, and proper DOI inclusion. These modifications are reflected in Section 5.2 (p. 6, lines 251–261), Section 5.5 (p. 9, lines 372–378), Section 6 (p. 10, lines 420–428), Section 7 (p. 11, lines 450–459), as well as on p. 14 (lines 618–619), p. 15 (lines 641–642, 652–654, 658, 665–666), p. 16 (lines 686–687), and p. 17 (lines 770–771).

 

Point 1: The English could be improved to more clearly express the research

We sincerely thank the Reviewer for this constructive comment regarding language quality. The manuscript has been thoroughly revised to enhance linguistic clarity, grammatical accuracy, and scientific precision. All sections have been carefully proofread, and phrasing has been optimized to ensure that the research objectives, methodology, and conclusions are expressed more clearly and concisely.

Should any further linguistic refinements be required, we intend to utilize the professional English editing services offered by MDPI Journals. At present, we are unable to engage these services in advance, as our current funding arrangement stipulates that the article processing charge (APC) and any additional editorial services will be covered within a single payment tranche. Upon acceptance of the manuscript, we will promptly proceed with the combined payment for both publication and language editing services to ensure the final version meets the journal's linguistic standards.

We remain fully committed to maintaining the highest editorial quality and are prepared to implement any additional linguistic improvements during the production stage as needed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

The  author has addressed all comments and suggestions as requested.