A Novel Bicistronic Adenovirus Vaccine Elicits Superior and Comprehensive Protection Against BVDV

1. Summary

Firstly, we would like to express our sincere thanks for your kind letter and the constructive comments provided by the reviewers on our article (Manuscript Number: microorganisms-4098028). These comments have been invaluable and have greatly contributed to the improvement of our manuscript. All authors have carefully considered each of the reviewers' suggestions. In response, we have made the necessary revisions to the manuscript in order to meet the requirements of your journal. Should any further revisions be necessary, we would be happy to address them and are grateful for your continued support. To facilitate the review process, all textual changes in the revised manuscript are highlighted using the following color code: Red: Revisions addressing Reviewer 1's comments. Green: Revisions addressing Reviewer 2's comments. Blue: Revisions addressing comments from both Reviewers. A point-by-point response to the reviewers' comments is provided below.

2. Point-by-point response to Comments and Suggestions for Authors

(1) The novelty of the study lies in the bicistronic adenoviral design combining antigens from different BVDV genotypes. This should be stated more clearly in the Introduction and Discussion, with a sharper distinction from previous single-antigen adenovirus or protein-based vaccine studies.

Response: We sincerely thank the reviewer for this valuable suggestion. We have now explicitly highlighted the novelty of our bicistronic adenovirus platform and its distinction from prior studies in both the Introduction and Discussion sections.

Introduction

Lines 57-61: However, most prior studies using adenoviral vectors or subunit platforms have focused on expressing single BVDV antigens [15-17, 21, 22]. The potential synergistic effects of combining these key antigens from different BVDV genotypes within a single bicistronic adenovirus vector to elicit broader and more potent immunity remain an area of active investigation.

Lines 62-65: In this study, we hypothesized that a novel bicistronic adenovirus vector co-expressing key antigenic proteins from BVDV-1 and BVDV-2 would elicit a superior protective immune response compared to conventional inactivated vaccines and pre-viously reported single-antigen platforms.

Discussion

Lines 354-359: While previous studies have utilized adenoviral vectors to express single BVDV anti-gens like E2 [21] or C [22], and protein-based vaccines focusing on E2 or E0 [15-17], our study advances the field by constructing and characterizing two novel bicistronic vectors co-expressing E0 and E2 from BVDV-1 or E2 proteins from both BVDV-1 and BVDV-2 genotypes. This design aims to harness potential synergistic effects for broad-er and more potent immunity.

Lines 370-381: The most direct evidence of our vaccine's enhanced immunogenicity in this model is reflected in the humoral response. The rAdV-I E2+II E2 vaccine induced neutralizing antibody (NAb) titers that were significantly higher and developed more rapidly than those elicited by the commercial inactivated vaccine. Moreover, the NAb levels achieved appeared comparable or superior to those reported for other BVDV subunit platforms, such as E2-based or virus-like particle (VLP) vaccines [17, 33, 34], which is consistent with the recognized capacity of adenoviral vectors to elicit humoral immunity. Notably, our bicistronic vaccines further modulated the cellular immune landscape, inducing distinct patterns of T-helper cell polarization. Specifically, we observed a mixed response from rAdV-I E0+I E2 versus a Th2-bias from rAdV-I E2+II E2. This suggests that the specific antigen combination within a bicistronic vector may actively influence immune polarization, a finding that merits further investigation to elucidate the underlying mechanisms.

(2) While the murine model is appropriate for proof-of-concept immunogenicity and protection studies, the Discussion should more clearly acknowledge the limitations of extrapolating these findings to cattle. Statements regarding vaccine superiority should be framed cautiously, emphasizing the need for validation in the natural host.

Response: We agree completely with the reviewer. The murine model serves as a robust proof-of-concept system, and findings must be validated in the target species. We have revised the Discussion to more cautiously frame our conclusions and explicitly state this critical limitation.

Lines 399-402: It is important to note that these promising results were obtained in a murine model. While they provide strong proof-of-concept, further evaluation in bovine hosts is es-sential and warranted to confirm their protective efficacy, safety, and broader applicability in the natural target species.

(3) Some redundancy is present across the Results and Discussion sections, particularly in the repeated description of immune response trends. The Results section could be streamlined by reducing narrative repetition already evident in figures. Similarly, the Discussion could be shortened by avoiding restatement of numerical outcomes.

Response: We thank the reviewer for pointing out the redundancy. We have carefully revised both the Results and Discussion sections to streamline the narrative.

Results

Lines 244-248: At both day 28 and day 42 post-immunization, the two recombinant adenovirus vac-cine groups and the inactivated vaccine group all elicited significantly higher numbers of IFN-γ⁺ SFCs compared to the NC group (p < 0.01; Figure 3A, B). Notably, the response induced by rAdV-I E0+I E2 was significantly stronger than that of the PC group at day 28 (p < 0.01).

Lines 276-280: Notably, the two adenoviral vectors elicited distinct immune polarization patterns. The rAdV-I E0+I E2 vaccine induced a mixed response, with Th1 bias at days 7, 14, and 42, but a Th2 bias at the intervening time points. In contrast, the rAdV-I E2+II E2 vaccine consistently promoted a Th2-dominant response (ratio <1), with only a brief, transient Th1 shift at day 21.

Lines 297-301: As shown in Figure 5, both rAdV-I E0+I E2 and rAdV-I E2+II E2 vaccines induced significantly higher neutralizing antibody levels than the NC at all timepoints (p < 0.01). Compared to the PC, both recombinant vaccines elicited significantly higher neutral-izing titers on days 14 and 42 (p < 0.05), while only the rAdV-I E2+II E2 vaccine maintained this superior response at day 28 (p < 0.01).

Lines 315-327: To evaluate vaccine protection, viral loads in blood and multiple tissues (heart, liver, spleen, lungs, kidneys, and small intestine) were quantified by RT-qPCR at 14 days post-BVDV infection. Compared to the negative control (NC), all vaccinated groups significantly suppressed viral replication. Mice immunized with rAdV-I E0+I E2 exhibited reduced viral loads in the spleen, lungs, kidneys, and small intestine (Figure 6D–G; p < 0.05). The rAdV-I E2+II E2 vaccine conferred superior efficacy, significantly lowering viral loads across all tissues examined (Figure 6A–G; p < 0.01). Notably, viral loads in the blood and spleen of this group were also lower than those in the positive control (PC) group (Figure 6A, D; p < 0.05). Furthermore, the commercial inactivated vaccine provided substantial protection, with significantly decreased viral loads in the heart, liver, lungs, kidneys, and small intestine relative to the NC group (Figure 6B–G; p < 0.05). Collectively, these results demonstrate that while all vaccines were effective, the rAdV-I E2+II E2 vaccine induced the most robust and comprehensive protection against BVDV replication.

Lines 354-388: While previous studies have utilized adenoviral vectors to express single BVDV antigens like E2 [21] or C [22], and protein-based vaccines focusing on E2 or E0 [15-17], our study advances the field by constructing and characterizing two novel bicistronic vectors co-expressing E0 and E2 from BVDV-1 or E2 proteins from both BVDV-1 and BVDV-2 genotypes. This design aims to harness potential synergistic effects for broader and more potent immunity. The successful packaging and confirmed genetic stability of our vaccines across serial passages are consistent with the known robustness of the adenovirus platform, but here we demonstrate this stability for more complex bicistronic constructs, ensuring consistent immunogen delivery.

The potent Th1-skewed cellular immune response elicited by our vaccines, particularly rAdV-I E0+I E2, aligns with the established capacity of adenoviral vectors to stimulate T-cell immunity. However, the sustained and significantly increasing IFN-γ production we observed between day 28 and 42 goes beyond the responses typically reported for inactivated vaccines or even some single-antigen adenovirus constructs [21, 22]. This underscores the potential of our bicistronic design to drive a more durable T-cell response, which is crucial for long-term protection against viral infections.

The most direct evidence of our vaccine's enhanced immunogenicity in this model is reflected in the humoral response. The rAdV-I E2+II E2 vaccine induced neutralizing antibody (NAb) titers that were significantly higher and developed more rapidly than those elicited by the commercial inactivated vaccine. Moreover, the NAb levels achieved appeared comparable or superior to those reported for other BVDV subunit platforms, such as E2-based or virus-like particle (VLP) vaccines [17, 33, 34], which is consistent with the recognized capacity of adenoviral vectors to elicit humoral immunity. Notably, our bicistronic vaccines further modulated the cellular immune landscape, inducing distinct patterns of T-helper cell polarization. Specifically, we observed a mixed response from rAdV-I E0+I E2 versus a Th2-bias from rAdV-I E2+II E2. This suggests that the specific antigen combination within a bicistronic vector may actively influence immune polarization, a finding that merits further investigation to elucidate the underlying mechanisms.

The comprehensive protection conferred by rAdV-I E2+II E2, resulting in reduced viral loads across all tissues, demonstrates a clear advantage. Previous challenge stud-ies using VLPs or inactivated vaccines have often shown partial protection, primarily reducing clinical signs but not always achieving significant viral clearance in all organs [24, 34]. In contrast, our rAdV-I E2+II E2 vaccine's ability to significantly lower viral loads in the blood and spleen below the level of the inactivated vaccine group sets a new benchmark in this murine model.

(4) The observed differences in Th1/Th2 polarization between the two recombinant vaccines are interesting and potentially important. However, the mechanistic basis for this divergence remains speculative and should be presented more cautiously or supported with additional references.

Response: We agree that the mechanistic insight is speculative. We have toned down the language in the Discussion to present this finding more cautiously as an observation that warrants future investigation.

Lines 376-381: Notably, our bicistronic vaccines further modulated the cellular immune landscape, inducing distinct patterns of T-helper cell polarization. Specifically, we observed a mixed response from rAdV-I E0+I E2 versus a Th2-bias from rAdV-I E2+II E2. This suggests that the specific antigen combination within a bicistronic vector may actively influence immune polarization, a finding that merits further investigation to elucidate the underlying mechanisms.

(5) The statistical methods are generally appropriate, but the manuscript should clarify the rationale for the selected post hoc tests and explicitly state the number of animals used per assay where subsampling was performed (e.g., ELISpot). This will improve transparency and reproducibility.

Response: We appreciate this suggestion to enhance methodological clarity. We have added the requested details to the Materials and Methods (Section 2.11) and the ELISpot assay section (2.5).

Lines 139-140: Briefly, splenocytes were isolated from three randomly selected mice per group.

Lines 202-207: Following a significant ANOVA result (p < 0.05), post hoc comparisons between relevant groups were performed to identify specific differences. A p-value < 0.05 was con-sidered statistically significant. The sample size (number of biological replicates, n) for each experiment is explicitly stated in the corresponding methods sections above. Viral loads in tissue samples and TCID₅₀ values were calculated separately using Microsoft Excel (Microsoft Corporation, USA).

1. Summary

Firstly, we would like to express our sincere thanks for your kind letter and the constructive comments provided by the reviewers on our article (Manuscript Number: microorganisms-4098028). These comments have been invaluable and have greatly contributed to the improvement of our manuscript. All authors have carefully considered each of the reviewers' suggestions. In response, we have made the necessary revisions to the manuscript in order to meet the requirements of your journal. Should any further revisions be necessary, we would be happy to address them and are grateful for your continued support. To facilitate the review process, all textual changes in the revised manuscript are highlighted using the following color code: Red: Revisions addressing Reviewer 1's comments. Green: Revisions addressing Reviewer 2's comments. Blue: Revisions addressing comments from both Reviewers. A point-by-point response to the reviewers' comments is provided below.

2. Point-by-point response to Comments and Suggestions for Authors