Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Vaccine Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Wang et al “Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Immunogenic and Vaccine Potential” describes the bioinformatics guided prediction of B cells epitopes on the nucleocapsid protein of canine papillomaviruses 1 and 2; the cloning and expression in E.coli of both CPV1 and CPV2 L1 proteins; the immunization of mice with the recombinant proteins and three selected peptides; and the evaluation of the humoral and cellular immune response against these antigens.

The manuscript is generally well written, and the mapping of three new conserved B cell epitopes on the L1 protein represents original findings. However, there are important limitations in the description of the methodology, as well as in the presentation and interpretation of the results, which must be addressed before publication.

1. Title: There is redundancy in the title. The phrase “…Vaccine potential” is sufficient; the word immunogenic is not required, since vaccine potential implies immunogenicity.
2. Page 3, lines 96-97. “Three conserved epitope peptides (VIVPKVS, TPSGSLV, and TVVDNTR) were synthesized and conjugated to bovine serum albumin (BSA) by standard solid-phase peptide synthesis”. The phrasing is incorrect; peptides should first be synthesized and subsequently conjugated to BSA. In addition, the conjugation procedure must be described.
3. Peptide length rationale: The choice of 7-mer peptides requires justification. Seven amino acid peptides are not optimal for sequential B cell epitopes, nor for MHC-I or MHC-II binding.
4. PAGE and WB: Please provide more methodological details in Materials and Methods, including acrylamide concentrations, molecular weight markers (with MW of all proteins), antibodies used, blocking procedure, transfer conditions, incubation times and temperatures, and the luminescent substrate employed. These details are essential for reproducibility.
5. Page 3, lines 125/126. “Purified CPV1 and CPV2 L1 proteins, as well as synthetic multi-epitope peptides, were emulsified with Freund’s complete adjuvant for primary immunization” The description of the immunization is unclear. It is stated earlier that peptides were conjugated to BSA, but here “multi-epitope peptides” are mentioned. This is contradictory. Please clarify how peptides were presented to the immune system.
6. Protein expression by WB. Figure 4. A negative control is required, such as non-induced E. coli cultures or non-recombinant E. coli grown in parallel. This is necessary to confirm that the band recognized by the anti-histidine mAb corresponds to the recombinant protein, since some E. coli proteins contain polyhistidine sequences.
7. Humoral immune response. For the indirect ELISAS in Figure 5 please specify which protein was used to coat the plate in each case. I imagine that CPV1 L1 was used for group 1 and CPV2 L1 for group 2, but for groups immunized with peptides, this information is missing.
8. Figure 5 caption and statistics: The statistical test applied must be declared. If ANOVA was used, normality and variance homogeneity should be tested and reported in Materials and Methods. For small sample sizes (n=5), non-parametric tests are more appropriate. Also, specify the post-hoc test applied. A Student’s t-test is not suitable when more than two groups are compared.
9. ELISA positivity criteria: Define in Materials and Methods the criteria for positivity (e.g., OD ≥ 2× average of negative control sera). This appears to be met for anti-protein sera but is less clear for anti-peptide sera at day 14.
10. Figure 5B titration curves: The titration data appear inconsistent. For example, CPV1 OD values at 10⁻⁶ and 10⁻⁷ are nearly identical, suggesting background noise rather than true signal. The same is observed for other titration curves with anti-protein sera. Proper titration should show a clear drop in OD with serial dilutions. This experiment should be repeated or the data excluded.
11. Protein characterization: The molecular weight of L1 proteins under non-reducing conditions should be also reported. Are they forming VLPs or only monomers? This is highly relevant to immunogenicity and must be analyzed.
12. ELISPOT (Figure 6): Clarify in Materials and Methods the number of cells per well and the positivity criteria. ELISPOT assays should include parallel wells with and without antigen to reduce background. The reported numbers of IFN-γ secreting cells are very low and do not convincingly demonstrate a T cell response, particularly against peptides. These results should be critically reinterpreted or removed.
13. Western blot (Figure 7 and Supplementary Information): Include molecular weights of all markers. Review carefully: the markers labeled 55 kDa and 70 kDa appear inconsistent between CPV1 and CPV2 antisera. Additionally, in peptide 1 sera, a faint band is observed in the CPV2 lane, and a similar band appears in the negative control lane, yet the rectangle obscures this. Non-specific signals must be acknowledged, and figures adjusted for clarity.

 Conclusions: While some humoral immune response against the peptides was demonstrated, it was:

• Much lower than that against the recombinant protein.
• Tested only in mice and not in the final species.
• The functionality of the antibodies (e.g., neutralizing capacity) remains undetermined.
• Cross-reactivity was stronger for proteins than for peptides.

Therefore, the conclusions should be moderated. The statement “this study provides a theoretical and experimental basis for the development of broadly protective CPV vaccines…” is too strong given the preliminary nature of the peptide data.

 

Author Response

Comments and Suggestions for Authors

The manuscript by Wang et al “Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Immunogenic and Vaccine Potential” describes the bioinformatics guided prediction of B cells epitopes on the nucleocapsid protein of canine papillomaviruses 1 and 2; the cloning and expression in E.coli of both CPV1 and CPV2 L1 proteins; the immunization of mice with the recombinant proteins and three selected peptides; and the evaluation of the humoral and cellular immune response against these antigens.

The manuscript is generally well written, and the mapping of three new conserved B cell epitopes on the L1 protein represents original findings. However, there are important limitations in the description of the methodology, as well as in the presentation and interpretation of the results, which must be addressed before publication.

R: We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the originality of identifying conserved cross-reactive B-cell epitopes in CPV1 and CPV2 L1 proteins. We also appreciate the constructive comments regarding methodological details, data presentation, and result interpretation. In response, we have revised the manuscript to improve clarity and rigor. Specifically, we added details on peptide conjugation and immunization procedures, corrected relevant text, and revised the Results and Discussion sections to present the findings more cautiously and avoid overinterpretation.

1. Title: There is redundancy in the title. The phrase “…Vaccine potential” is sufficient; the word immunogenic is not required, since vaccine potential implies immunogenicity.

R: We thank the reviewer for this helpful suggestion. We agree that the original title contained redundant wording, as “vaccine potential” already implies immunogenic relevance. Accordingly, we have revised the title for greater conciseness and precision.

Original title: Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Immunogenic and Vaccine Potential

Revised title: Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Vaccine Potential.

2. Page 3, lines 96-97. “Three conserved epitope peptides (VIVPKVS, TPSGSLV, and TVVDNTR) were synthesized and conjugated to bovine serum albumin (BSA) by standard solid-phase peptide synthesis”. The phrasing is incorrect; peptides should first be synthesized and subsequently conjugated to BSA. In addition, the conjugation procedure must be described.

R: We thank the reviewer for this important comment. We agree that peptide synthesis and BSA conjugation are distinct steps and should be described separately. We have revised the text for accuracy and added details on peptide conjugation to the Materials and Methods section.

Revised text: Three conserved epitope peptides (VIVPKVS, TPSGSLV, and TVVDNTR) were synthesized by standard solid-phase peptide synthesis (GenScript Biotech, Nanjing, China) with >95% purity. To enhance immunogenicity, a terminal cysteine residue was added during synthesis, and the peptides were conjugated to bovine serum albumin (BSA) using maleimide-mediated coupling according to the manufacturer’s protocol. The conjugated peptides were purified by HPLC and verified by mass spectrometry before use.

3. Peptide length rationale: The choice of 7-mer peptides requires justification. Seven amino acid peptides are not optimal for sequential B cell epitopes, nor for MHC-I or MHC-II binding.

R: We thank the reviewer for this important comment. We agree that the rationale for selecting 7-mer peptides should be clearly explained, as short peptides may have limited immunogenicity and are not optimal for T-cell epitope presentation.

These peptides were selected based on integrated linear B-cell epitope prediction and conservation analysis of CPV1 and CPV2 L1 proteins. Our goal was to identify minimal conserved linear motifs that may function as cross-reactive B-cell antigenic determinants, rather than to define optimized T-cell epitopes or vaccine-ready peptide antigens. We have clarified this rationale and added the limitation to the Discussion.

 

Added text: “Although 7-mer peptides are shorter than the lengths generally considered optimal for robust B-cell or T-cell immunogenicity, they were selected to identify minimal conserved linear motifs shared by CPV1 and CPV2 L1 proteins. This design aimed to define potential cross-reactive antigenic regions rather than optimized vaccine peptides. Future studies using longer peptides, flanking residues, improved carriers, or multivalent display platforms may enhance immunogenicity and better reflect naturally processed or conformationally relevant epitopes.”

4. PAGE and WB: Please provide more methodological details in Materials and Methods, including acrylamide concentrations, molecular weight markers (with MW of all proteins), antibodies used, blocking procedure, transfer conditions, incubation times and temperatures, and the luminescent substrate employed. These details are essential for reproducibility.

R: We thank the reviewer for this constructive comment. We agree that additional PAGE and Western blot details are needed to improve reproducibility. We have therefore expanded the Materials and Methods section to include gel concentration, molecular weight marker, antibody information, blocking conditions, transfer parameters, incubation conditions, detection reagent, and negative controls.

Revised text: Protein samples were separated by SDS-PAGE on 10% polyacrylamide gels under reducing conditions, alongside a prestained protein molecular weight marker. Proteins were transferred onto PVDF membranes using a semi-dry transfer system at 20 V for 40 min. Membranes were blocked with 5% skim milk in PBST for 1 h at room temperature and incubated overnight at 4 °C with mouse anti-His monoclonal antibody (1:5000). After washing, membranes were incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) for 1 h at room temperature. Signals were detected using enhanced chemiluminescence substrate (Thermo Fisher Scientific, USA). Non-induced E. coli BL21 cultures and empty vector-transformed bacteria were included as negative controls.

5. Page 3, lines 125/126.“Purified CPV1 and CPV2 L1 proteins, as well as synthetic multi-epitope peptides, were emulsified with Freund’s complete adjuvant for primary immunization” The description of the immunization is unclear. It is stated earlier that peptides were conjugated to BSA, but here “multi-epitope peptides” are mentioned. This is contradictory. Please clarify how peptides were presented to the immune system.

R: We thank the reviewer for this constructive comment. We agree that the original wording was ambiguous regarding the peptide form used for immunization. In the revised manuscript, we have clarified that the immunizing peptides were BSA-conjugated synthetic peptides, not unconjugated “multi-epitope peptides.” Accordingly, the term “multi-epitope peptides” has been removed throughout the manuscript to ensure consistency with the Materials and Methods.

 

Revised text: “BSA-conjugated synthetic peptides and purified recombinant CPV1/CPV2 L1 proteins were emulsified with Freund’s complete adjuvant for primary immunization.”

6. Protein expression by WB. Figure 4. A negative control is required, such as non-induced E. coli cultures or non-recombinant E. coli grown in parallel. This is necessary to confirm that the band recognized by the anti-histidine mAb corresponds to the recombinant protein, since some E. coli proteins contain polyhistidine sequences.

R: We thank the reviewer for this important comment. We agree that additional negative controls, such as non-induced recombinant E. coli cultures or non-recombinant E. coli lysates, would further strengthen the Western blot analysis.

In this study, recombinant protein identity was supported by the expected molecular weight, inducible expression pattern on SDS-PAGE, and anti-His immunoblot reactivity in purified protein fractions. However, we acknowledge that the absence of the suggested lysate controls limits our ability to fully exclude nonspecific anti-His reactivity in crude bacterial lysates. We have therefore revised the manuscript to clarify this point and avoid overstating the specificity of the Western blot results.

Revised text: “Protein identity was supported by the expected molecular weight, inducible expression pattern, and anti-His immunoblot reactivity of the purified recombinant proteins. However, because non-induced and non-recombinant E. coli lysate controls were not included in the original Western blot experiment, the possibility of background reactivity in crude bacterial lysates cannot be completely excluded.”

We have also noted this as a methodological limitation. Nevertheless, the prominent induced band at the expected size, together with anti-His reactivity in the purified fraction, supports that the detected band corresponds predominantly to the recombinant L1 protein.

7. Humoral immune response. For the indirect ELISAS in Figure 5 please specify which protein was used to coat the plate in each case. I imagine that CPV1 L1 was used for group 1 and CPV2 L1 for group 2, but for groups immunized with peptides, this information is missing.

R: We thank the reviewer for pointing out this omission. We agree that the coating antigens used in the indirect ELISA should be clearly specified. We have revised the Materials and Methods section and figure legend accordingly.

Revised text: For indirect ELISA, plates were coated with purified recombinant CPV1 L1 or CPV2 L1 protein, depending on the immunization group and assay purpose. Sera from mice immunized with recombinant CPV1 L1 or CPV2 L1 were tested against the corresponding homologous L1 protein. For peptide-immunized groups, plates were coated with recombinant CPV1 and/or CPV2 L1 proteins to determine whether peptide-induced antibodies recognized the recombinant L1 antigens and showed cross-reactive binding.

8. Figure 5 caption and statistics: The statistical test applied must be declared. If ANOVA was used, normality and variance homogeneity should be tested and reported in Materials and Methods. For small sample sizes (n=5), non-parametric tests are more appropriate. Also, specify the post-hoc test applied. A Student’s t-test is not suitable when more than two groups are compared.

R: We thank the reviewer for this helpful suggestion. We agree that non-parametric analysis is more appropriate for Figure 5 given the small sample size (n = 5 per group). We have revised the statistical analysis and clarified the methods in both the Materials and Methods section and Figure 5 legend. Specifically, multiple-group comparisons in Figure 5 were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. We also corrected the text to avoid implying that Student’s t-test was used for comparisons among more than two groups.

Revised text: For datasets with relatively small sample sizes, including those shown in Figure 5, statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. Comparisons between two groups were performed using Student’s t-test. A p value < 0.05 was considered statistically significant.

Revised Figure 5 legend: Figure 5. Humoral immune responses induced by recombinant CPV1/CPV2 L1 proteins and peptide immunization. Indirect ELISA was performed to evaluate antigen-specific IgG responses in sera from immunized mice. Plates were coated with purified recombinant CPV1 L1 or CPV2 L1 protein, as indicated. Data are shown as mean ± SD (n = 5 per group). Statistical significance among groups was analyzed using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. A p value < 0.05 was considered statistically significant.

9. ELISA positivity criteria: Define in Materials and Methods the criteria for positivity (e.g., OD ≥ 2× average of negative control sera). This appears to be met for anti-protein sera but is less clear for anti-peptide sera at day 14.

R: We appreciate the reviewer’s helpful suggestion. We have now defined the ELISA positivity criterion in the Materials and Methods section.

Revised text: “Samples were considered ELISA-positive when the OD450 value was at least twofold higher than the mean OD450 value of the negative control sera.”

Based on this criterion, anti-protein sera showed clear positive responses, whereas some anti-peptide sera at day 14 were weaker and close to the positivity threshold. We have revised the Results section to describe these early anti-peptide responses more cautiously.

10. Figure 5B titration curves: The titration data appear inconsistent. For example, CPV1 OD values at 10⁻⁶ and 10⁻⁷ are nearly identical, suggesting background noise rather than true signal. The same is observed for other titration curves with anti-protein sera. Proper titration should show a clear drop in OD with serial dilutions. This experiment should be repeated or the data excluded.

R: We thank the reviewer for carefully examining the ELISA titration curves. We agree that some OD450 values at the highest serum dilutions, particularly 10⁻⁶ and 10⁻⁷, were close to the negative-control background and therefore may not reliably represent true antibody-specific reactivity.

Because these terminal dilution points were outside the informative range of the assay, we have revised Figure 5B to present the interpretable dilution range and have avoided overinterpretation of background-level signals. In the revised analysis, antibody reactivity was interpreted according to the ELISA positivity criterion defined in the Materials and Methods section. Signals approaching the negative-control background were not considered evidence of meaningful antibody reactivity.

The revised Figure 5B more clearly shows that recombinant L1 protein-immunized sera retained stronger reactivity across serial dilutions than peptide-immunized sera, whereas peptide-induced responses were weaker and declined more rapidly.

 

11. Protein characterization: The molecular weight of L1 proteins under non-reducing conditions should be also reported. Are they forming VLPs or only monomers? This is highly relevant to immunogenicity and must be analyzed.

R: We thank the reviewer for this important comment. We agree that the native structural state of papillomavirus L1 proteins, including whether they form monomers, capsomeres, oligomers, or VLPs, is highly relevant to immunogenicity.

In this study, CPV1 and CPV2 L1 proteins were expressed in a prokaryotic system and used as recombinant L1 protein antigens for immunogenicity and epitope-reactivity analysis. We did not experimentally demonstrate VLP formation. Therefore, to avoid overinterpretation, we have revised the manuscript to consistently describe them as recombinant CPV1/CPV2 L1 proteins or antigens, rather than VLPs.

We also clarified that the molecular weights reported were based on denaturing SDS-PAGE/Western blot analysis and were used to confirm recombinant protein expression, not native oligomeric status. Because non-reducing SDS-PAGE, native PAGE, SEC, DLS, or TEM analyses were not performed, this point has been added as a limitation.

Revised text: “Papillomavirus L1 proteins have intrinsic self-assembly potential; however, prokaryotic expression does not necessarily indicate complete VLP formation. Previous studies showed that bacterially expressed papillomavirus L1 proteins may form pentameric capsomere-like or other oligomeric structures while retaining antigenicity and immunogenicity. In this study, recombinant CPV1 and CPV2 L1 proteins were used as protein antigens, and no direct structural evidence was obtained to confirm VLP formation. Therefore, we avoided describing these proteins as VLPs. Further structural analyses, such as non-reducing SDS-PAGE, native PAGE, SEC, DLS, or TEM, will be needed to determine whether they form capsomeres, higher-order oligomers, or VLP-like particles.”

 

12. ELISPOT (Figure 6): Clarify in Materials and Methods the number of cells per well and the positivity criteria. ELISPOT assays should include parallel wells with and without antigen to reduce background. The reported numbers of IFN-γ secreting cells are very low and do not convincingly demonstrate a T cell response, particularly against peptides. These results should be critically reinterpreted or removed.

R: We thank the reviewer for this critical and constructive comment. We agree that the ELISpot methodology and interpretation required clarification. We have revised the Materials and Methods section to specify the number of splenocytes per well, antigen stimulation conditions, unstimulated background-control wells, background subtraction, and positivity criteria.

Revised Materials and Methods text: “Splenocytes were seeded at [insert cell number] cells/well in IFN-γ ELISpot plates and stimulated with recombinant CPV1/CPV2 L1 proteins or synthetic peptides at [insert antigen concentration]. Parallel wells without antigen were included as background controls. IFN-γ spot-forming cells were developed and counted according to the manufacturer’s instructions. Antigen-specific responses were calculated by subtracting spots in unstimulated wells from those in antigen-stimulated wells. A response was considered positive when spot numbers were above background and at least twofold higher than the corresponding unstimulated control.”

We agree that the IFN-γ spot numbers were low, especially after peptide stimulation. Therefore, we have revised the Results and Discussion to avoid overinterpretation and now describe these data as “modest antigen-associated cellular responses” rather than strong T-cell responses.

Revised Results text: “IFN-γ ELISpot analysis showed detectable but relatively low numbers of IFN-γ-secreting cells after stimulation with recombinant L1 proteins or selected peptides. After background subtraction, these responses were interpreted as modest antigen-associated cellular responses. Peptide-induced responses were weaker than those induced by recombinant L1 proteins and should be interpreted cautiously.”

Revised Discussion text: “Although IFN-γ ELISpot analysis suggested modest antigen-associated cellular activation, the numbers of IFN-γ-secreting cells were low, particularly after peptide stimulation. Thus, these data do not provide strong evidence for robust T-cell epitope activity. Since this study mainly focused on conserved cross-reactive B-cell epitopes, the ELISpot results should be considered supportive but limited. Further studies using optimized peptide pools, larger sample sizes, intracellular cytokine staining, T-cell proliferation assays, and flow cytometry will be needed to define CPV1/CPV2 L1-specific cellular responses more rigorously.”

 

13. Western blot (Figure 7 and Supplementary Information): Include molecular weights of all markers. Review carefully: the markers labeled 55 kDa and 70 kDa appear inconsistent between CPV1 and CPV2 antisera. Additionally, in peptide 1 sera, a faint band is observed in the CPV2 lane, and a similar band appears in the negative control lane, yet the rectangle obscures this. Non-specific signals must be acknowledged, and figures adjusted for clarity.

R: We thank the reviewer for this careful evaluation. We have re-examined the original Western blot images and revised Figure 7 and the Supplementary Information accordingly. Molecular weights for all markers have now been included, and the inconsistent 55 kDa and 70 kDa labels between the CPV1 and CPV2 antisera panels have been corrected.

We also revised the figure layout by removing/replacing rectangles that obscured faint bands. We agree that the faint band in the CPV2 lane probed with peptide 1 serum is similar to the weak signal in the negative-control lane. Therefore, this signal is now acknowledged as likely background or non-specific reactivity and is no longer interpreted as clear peptide-specific cross-reactivity.

The Results, Discussion, and Figure 7 legend have been revised to clarify that only bands clearly stronger than the negative-control background were considered specific antigen-reactive signals. Weak bands comparable to the negative control were interpreted cautiously as non-specific background.

 

 Conclusions: While some humoral immune response against the peptides was demonstrated, it was:

• Much lower than that against the recombinant protein.
• Tested only in mice and not in the final species.
• The functionality of the antibodies (e.g., neutralizing capacity) remains undetermined.
• Cross-reactivity was stronger for proteins than for peptides.

Therefore, the conclusions should be moderated. The statement “this study provides a theoretical and experimental basis for the development of broadly protective CPV vaccines…” is too strong given the preliminary nature of the peptide data.

R: We thank the reviewer for this important comment. We agree that the original conclusion was too strong given the preliminary nature of the peptide-immunization data. We have therefore moderated the conclusions in the Abstract, Discussion, and Conclusion sections.

In the revised manuscript, we clarify that although selected peptides induced detectable humoral responses in mice, these responses were weaker than those induced by recombinant L1 proteins. We also acknowledge that the peptide responses were tested only in mice, not in the target canine host, and that antibody functionality, including neutralizing activity, was not evaluated. In addition, peptide-induced cross-reactivity was weaker and less consistent than that observed with anti-protein sera.

Accordingly, the original statement has been replaced with the following more cautious wording:

Revised text: “This study provides preliminary immunological evidence and candidate epitope information that may support future rational design of CPV L1-based vaccines, but further validation in dogs, including neutralization assays and protective-efficacy studies, will be required.”

We also added a limitation statement emphasizing that these peptides should be considered candidate B-cell epitopes, not confirmed protective vaccine antigens, at this stage.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Manuscript Title: Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Immunogenic and Vaccine Potential

 

The study focuses on the bioinformatics-driven prediction of conserved linear B-cell epitopes within the L1 proteins of Canine Papillomavirus types 1 and 2 (CPV1 and CPV2), validating their immunogenicity through recombinant proteins and synthetic peptides in a mouse model.

Below is the detailed evaluation by section:

1. Introduction

• Strengths: The introduction defines the problem very well, highlighting the clinical relevance of CPV and the lack of commercial prophylactic vaccines. The strategy of targeting conserved epitopes to generate cross-immunity is well-justified.
• Needs Improvement: The authors do not justify why they focused exclusively on linear epitopes. It is well-established that in papillomaviruses (such as HPV), the primary protective and neutralizing immune response is generated against conformational epitopes present on the three-dimensional structure of the capsid (Virus-Like Particles - VLPs). The absence of this initial discussion weakens the study's premise.

2. Material and Methods

• Strengths: The computational screening pipeline was excellently designed, integrating multiple tools (AlphaFold, ABCpred, ConSurf) to ensure the selection of non-toxic and highly conserved sequences.
• Critical Flaws and Needs Improvement:
  • L1 Characterization: The authors expressed the L1 protein in E. coli. However, there is no assay (such as electron microscopy) to verify whether these purified proteins successfully self-assembled into Virus-Like Particles (VLPs). This is a critical flaw, as unassembled L1 proteins have a significantly inferior antigenic profile compared to assembled capsids.
  • Cellular Assay (ELISpot): The use of extremely short peptides, containing only 7 amino acids (e.g., VIVPKVS or TPSGSLV), without the inclusion of a T-helper epitope, is biologically inadequate for inducing T-cell activation in vitro in an ELISpot assay.

3. Results

• Strengths: The Western Blot assay (Figure 7) serves as a solid proof-of-concept, showing that antibodies generated against epitopes 2 and 3 can recognize both CPV1 and CPV2 L1 proteins, thus demonstrating cross-reactivity.
• Critical Flaws and Needs Improvement:
  • Overinterpretation of ELISpot Data (Figure 6): The authors claim in the text that the peptides "contribute to T-cell activation". However, Figure 6 and the text itself indicate that there was no statistically significant difference in IFN-\gamma secretion compared to the negative control (NC) group. The numerical data simply do not support the stated conclusion.
  • Lack of a Neutralization Assay: Demonstrating that an antibody binds to the L1 protein in an ELISA or Western Blot does not guarantee that it will neutralize a live viral infection. The authors admit this, but its absence severely limits any claims regarding true "vaccine potential."

4. Discussion

• Strengths: The authors are transparent in acknowledging the limitations of the murine model and the need for future viral neutralization assays. The suggestion to incorporate the discovered epitopes into VLP platforms is highly coherent.
• Needs Improvement: The discussion needs to temper its optimism. The antibody levels generated by the linear peptides were drastically lower than those induced by the full-length protein. The discussion should emphasize that these linear epitopes will likely only be useful if combined into larger, optimized constructs (multi-epitope platforms).

5. Statistical Analysis

• Needs Improvement: The authors report the use of ANOVA and Student's t-test. However, with a small sample size (n=5 mice per group), it is essential to state in section 2.9 whether the data were previously subjected to normality tests (e.g., Shapiro-Wilk) and homogeneity of variance tests, which are basic prerequisites for utilizing ANOVA.

6. Bibliographic References

• The references are up-to-date (several from 2024 and 2025) and well-formatted according to the journal's standards, adequately covering papillomavirus literature and bioinformatics tools. No major flaws were identified here, assuming the journal's 2026 publication format validates the timeline of the citations.

7. Bioethics

• Needs Improvement (Date Inconsistency): The Institutional Review Board Statement cites the approval as: "Approval No. 2026-33, Mar 11, 2024". A protocol physically approved in March 2024 should not receive a sequential numbering starting with "2026". The authors must correct this typo.
• Animal Welfare: Freund's Complete Adjuvant (FCA) was used for the primary immunization. FCA is known to cause severe local adverse reactions (such as granulomas and ulcers). It is absolutely necessary to include a sentence in the methods section detailing the clinical monitoring and the humane endpoint criteria adopted to manage these potential adverse reactions.

8. Mention of GPS

• None. After a rigorous analysis, the manuscript makes no mention, direct or indirect, of GPS (Global Positioning System). This is completely normal and expected, given that this is molecular, cellular, and animal model laboratory research with no geographic or ecological tracking components.

9. English Language Quality

• The English language presents flaws and requires mild to moderate improvement. While the text is generally fluid and logically structured for a scientific article, there are grammatical and agreement errors that a native copy-editor would easily catch.
• Examples of flaws in the text:
  • At the beginning of the discussion (line 360): "CPVs are increasingly recognized as a clinically relevant pathogens...". Using the singular article "a" with the plural noun "pathogens" is a clear grammatical error.
  • On line 369: "For a structural perspective...". The correct phrasing in academic English to start this sentence would be "From a structural perspective...".
  • On line 281: "maintained baseline level". Ideally, this should be pluralized to "baseline levels".
• Recommendation to the Editor/Authors: The manuscript's English is good enough for scientific comprehension but requires professional copy-editing (English proofreading) by a native speaker to correct syntax and agreement errors prior to publication.

The article is interesting and introduces novel peptide targets for CPV. However, I recommend a Major Revision. The authors need to adjust the forced interpretation of the ELISpot data, better justify the exclusive use of linear antigens, detail the statistical assumptions, correct the ethics protocol date, and undergo a thorough English grammar review.

Comments on the Quality of English Language

• The English language presents flaws and requires mild to moderate improvement. While the text is generally fluid and logically structured for a scientific article, there are grammatical and agreement errors that a native copy-editor would easily catch.
• Examples of flaws in the text:
  • At the beginning of the discussion (line 360): "CPVs are increasingly recognized as a clinically relevant pathogens...". Using the singular article "a" with the plural noun "pathogens" is a clear grammatical error.
  • On line 369: "For a structural perspective...". The correct phrasing in academic English to start this sentence would be "From a structural perspective...".
  • On line 281: "maintained baseline level". Ideally, this should be pluralized to "baseline levels".
• Recommendation to the Editor/Authors: The manuscript's English is good enough for scientific comprehension but requires professional copy-editing (English proofreading) by a native speaker to correct syntax and agreement errors prior to publication.

Author Response

Comments and Suggestions for Authors

 Manuscript Title: Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Immunogenic and Vaccine Potential

 The study focuses on the bioinformatics-driven prediction of conserved linear B-cell epitopes within the L1 proteins of Canine Papillomavirus types 1 and 2 (CPV1 and CPV2), validating their immunogenicity through recombinant proteins and synthetic peptides in a mouse model.

R: We thank the reviewer for the concise and accurate summary of our study. We appreciate the recognition that our work combines bioinformatics prediction with mouse-based experimental validation to identify conserved cross-reactive linear B-cell epitopes in CPV1 and CPV2 L1 proteins. We have carefully addressed all comments and revised the manuscript to improve clarity, methodological rigor, and overall presentation.

 

Below is the detailed evaluation by section:

1. Introduction

• Strengths:The introduction defines the problem very well, highlighting the clinical relevance of CPV and the lack of commercial prophylactic vaccines. The strategy of targeting conserved epitopes to generate cross-immunity is well-justified.
• Needs Improvement:The authors do not justify why they focused exclusively on linear epitopes. It is well-established that in papillomaviruses (such as HPV), the primary protective and neutralizing immune response is generated against conformational epitopes present on the three-dimensional structure of the capsid (Virus-Like Particles - VLPs). The absence of this initial discussion weakens the study's premise.

R: We thank the reviewer for this important comment. We agree that conformational epitopes displayed on native papillomavirus L1 capsids, particularly VLPs, are the major targets of potent neutralizing antibodies and the basis of current prophylactic vaccines. We have revised the Introduction to explicitly acknowledge this point and to better justify our focus on conserved linear epitopes.

In the revised manuscript, we clarify that this study does not aim to replace VLP-based approaches, but to identify conserved linear B-cell epitopes that may be useful for cross-reactive antigen design, immunodiagnostics, or future multi-epitope/complementary vaccine platforms. Linear epitopes were emphasized because they are suitable for conservation analysis across CPV genotypes and adaptable to synthetic peptide-based design.

Added text: “Although conformational epitopes presented on native L1 virus-like particles (VLPs) are the principal targets of potent neutralizing antibodies in papillomaviruses and form the basis of current prophylactic vaccines, conserved linear epitopes may still be useful for cross-reactive antigen design, immunodiagnostic development, and incorporation into optimized multi-epitope or complementary vaccine platforms.”

These revisions better position the study within papillomavirus immunology and clarify the rationale for focusing on conserved linear epitopes.

 

2. Material and Methods

• Strengths:The computational screening pipeline was excellently designed, integrating multiple tools (AlphaFold, ABCpred, ConSurf) to ensure the selection of non-toxic and highly conserved sequences.
• Critical Flaws and Needs Improvement:
  • L1 Characterization:The authors expressed the L1 protein in  coli. However, there is no assay (such as electron microscopy) to verify whether these purified proteins successfully self-assembled into Virus-Like Particles (VLPs). This is a critical flaw, as unassembled L1 proteins have a significantly inferior antigenic profile compared to assembled capsids.
  • Cellular Assay (ELISpot):The use of extremely short peptides, containing only 7 amino acids (e.g., VIVPKVS or TPSGSLV), without the inclusion of a T-helper epitope, is biologically inadequate for inducing T-cell activation in vitro in an ELISpot assay.

R: We thank the reviewer for these important comments and for recognizing the strength of our computational screening strategy. We agree that two points required clearer qualification.

First, although recombinant CPV1 and CPV2 L1 proteins were expressed and purified, VLP assembly was not directly confirmed by structural analyses such as TEM. We have therefore clarified that this study does not represent a formal characterization of assembled VLPs and have added this as a limitation.

Second, we agree that the 7-mer peptides are not optimal for rigorous evaluation of T-cell activation in ELISpot assays, especially without helper epitopes. Our aim was to identify minimal conserved linear B-cell motifs rather than optimized T-cell epitopes. Accordingly, we have moderated the interpretation of the ELISpot data and now describe them as preliminary evidence of antigen-associated IFN-γ responses, not definitive proof of peptide-specific T-cell epitope activity.

 

We also added a statement in the Discussion that future studies should use longer peptide constructs, improved antigen-presentation strategies, and more rigorous cellular immune assays to better evaluate CPV1/CPV2 L1-specific T-cell responses.

 

3. Results

• Strengths: The Western Blot assay (Figure 7) serves as a solid proof-of-concept, showing that antibodies generated against epitopes 2 and 3 can recognize both CPV1 and CPV2 L1 proteins, thus demonstrating cross-reactivity.
• Critical Flaws and Needs Improvement:

• Overinterpretation of ELISpot Data (Figure 6):The authors claim in the text that the peptides "contribute to T-cell activation". However, Figure 6 and the text itself indicate that there was no statistically significant difference in IFN-\gamma secretion compared to the negative control (NC) group. The numerical data simply do not support the stated conclusion.
• Lack of a Neutralization Assay:Demonstrating that an antibody binds to the L1 protein in an ELISA or Western Blot does not guarantee that it will neutralize a live viral infection. The authors admit this, but its absence severely limits any claims regarding true "vaccine potential."

R: We thank the reviewer for these constructive comments and for recognizing the value of the Western blot data showing cross-reactive recognition of CPV1 and CPV2 L1 proteins by antisera against epitopes 2 and 3. We agree that the ELISpot interpretation and vaccine-related claims required moderation.

(1) ELISpot interpretation: We agree that the original wording overstated the ELISpot results. Because peptide-immunized groups did not show statistically significant increases in IFN-γ secretion compared with the negative control, these data do not support robust peptide-specific T-cell activation. We have revised the Results and Discussion to interpret these findings cautiously as limited, non-significant IFN-γ responses under the current conditions.

Revised text: “Although peptide-immunized groups showed detectable IFN-γ spot formation in the ELISpot assay, these responses were not statistically significant compared with the negative control group. Therefore, the present data do not provide sufficient evidence to conclude that the tested short peptides directly induced robust T-cell activation.”

 

(2) Lack of neutralization assay: We agree that ELISA and Western blot binding do not demonstrate antibody neutralization. The absence of live-virus or pseudovirus neutralization assays is now explicitly acknowledged as a limitation. We have revised the manuscript to clarify that our data support conserved cross-reactive B-cell epitope identification and preliminary immunoreactivity, but not protective or neutralizing efficacy.

Added text: “While the identified epitopes demonstrated cross-reactive antibody recognition of CPV1 and CPV2 L1 proteins, the present study did not include live-virus or pseudovirus neutralization assays. Therefore, these findings should be interpreted as preliminary evidence of antigenic and immunoreactive potential rather than direct proof of neutralizing activity or protective vaccine efficacy.”

These revisions better align the conclusions with the experimental evidence and improve the rigor and balance of the manuscript.

 

4. Discussion

• Strengths: The authors are transparent in acknowledging the limitations of the murine model and the need for future viral neutralization assays. The suggestion to incorporate the discovered epitopes into VLP platforms is highly coherent.
• Needs Improvement: The discussion needs to temper its optimism. The antibody levels generated by the linear peptides were drastically lower than those induced by the full-length protein. The discussion should emphasize that these linear epitopes will likely only be useful if combined into larger, optimized constructs (multi-epitope platforms).

R: We thank the reviewer for this valuable suggestion. We agree that the Discussion should interpret peptide immunogenicity more cautiously. In the revised manuscript, we now acknowledge that antibody responses induced by the short linear peptides were much weaker than those elicited by full-length recombinant L1 proteins.

We further clarify that these conserved linear epitopes are unlikely to function effectively as standalone immunogens, but may be more useful when incorporated into optimized antigen designs, such as multi-epitope constructs, carrier-assisted formulations, or VLP-display platforms.

Added text: “Although the identified conserved linear epitopes showed cross-reactive immunoreactivity, antibody responses induced by individual short peptides were substantially weaker than those elicited by full-length recombinant L1 proteins. This suggests that these minimal linear motifs are unlikely to function as effective standalone immunogens. Their potential utility may instead depend on incorporation into larger optimized antigen constructs, such as multi-epitope platforms, carrier-assisted formulations, or VLP-display systems. In addition, short peptides may not efficiently engage multiple components of adaptive immunity without optimized delivery systems, molecular adjuvants, or multivalent antigen-presentation strategies.”

 

5. Statistical Analysis

• Needs Improvement: The authors report the use of ANOVA and Student's t-test. However, with a small sample size (n=5 mice per group), it is essential to state in section 2.9 whether the data were previously subjected to normality tests (e.g., Shapiro-Wilk) and homogeneity of variance tests, which are basic prerequisites for utilizing ANOVA.

R: We thank the reviewer for this important methodological comment. We agree that, given the small sample size (n = 5 per group), statistical assumptions should be clearly stated. We have revised Section 2.9 to specify that normality and variance homogeneity were assessed before parametric testing. One-way ANOVA with Tukey’s post hoc test was used only when these assumptions were met; otherwise, Kruskal–Wallis analysis with Dunn’s multiple-comparisons test was applied.

Revised text: “All experiments were performed using at least three independent biological replicates. Quantitative data are presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 9.0. Normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene’s test before parametric analysis. For multiple-group comparisons, one-way ANOVA followed by Tukey’s post hoc test was used when normality and equal variance assumptions were met; otherwise, the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test was applied. Comparisons between two groups were performed using Student’s t-test. A p value < 0.05 was considered statistically significant.”

 

6. Bibliographic References

• The references are up-to-date (several from 2024 and 2025) and well-formatted according to the journal's standards, adequately covering papillomavirus literature and bioinformatics tools. No major flaws were identified here, assuming the journal's 2026 publication format validates the timeline of the citations.

R: We thank the reviewer for the positive assessment of the references. We have carefully rechecked the reference list to ensure accuracy, consistency, and compliance with the journal’s formatting requirements.

 

7. Bioethics

• Needs Improvement (Date Inconsistency): The Institutional Review Board Statement cites the approval as: "Approval No. 2026-33, Mar 11, 2024". A protocol physically approved in March 2024 should not receive a sequential numbering starting with "2026". The authors must correct this typo.
• Animal Welfare: Freund's Complete Adjuvant (FCA) was used for the primary immunization. FCA is known to cause severe local adverse reactions (such as granulomas and ulcers). It is absolutely necessary to include a sentence in the methods section detailing the clinical monitoring and the humane endpoint criteria adopted to manage these potential adverse reactions.

R: We thank the reviewer for this important comment. The inconsistency in the ethics approval number and date was a typographical error and has been corrected. We also agree that animal welfare monitoring should be described more clearly, particularly given the use of Freund’s Complete Adjuvant. Accordingly, we have added details on daily post-immunization monitoring and humane endpoint criteria to the Materials and Methods section.

Revised text: “Animals were monitored daily after immunization for general health, injection-site reactions, mobility, behavior, and body weight. Humane endpoint criteria included severe local inflammation, impaired mobility, abnormal behavior, marked distress, or body weight loss exceeding 20%. The ethics approval number and approval date have been corrected in the revised manuscript.”.

 

8. Mention of GPS

• None. After a rigorous analysis, the manuscript makes no mention, direct or indirect, of GPS (Global Positioning System). This is completely normal and expected, given that this is molecular, cellular, and animal model laboratory research with no geographic or ecological tracking components.

R: We thank the reviewer for this clarification. GPS-related information is not applicable to this study, as the work was conducted entirely in a laboratory setting, including bioinformatics analysis, recombinant protein expression, mouse immunization, and immunological evaluation. No geographic tracking, field sampling, or spatial analysis was involved.

 

9. English Language Quality

• The English language presents flaws and requires mild to moderate improvement. While the text is generally fluid and logically structured for a scientific article, there are grammatical and agreement errors that a native copy-editor would easily catch.
• Examples of flaws in the text:

• At the beginning of the discussion (line 360): "CPVs are increasingly recognized as aclinically relevant pathogens...". Using the singular article "a" with the plural noun "pathogens" is a clear grammatical error.
• On line 369: "Fora structural perspective...". The correct phrasing in academic English to start this sentence would be "From a structural perspective...".
• On line 281: "maintained baseline level". Ideally, this should be pluralized to "baseline levels".

• Recommendation to the Editor/Authors: The manuscript's English is good enough for scientific comprehension but requires professional copy-editing (English proofreading) by a native speaker to correct syntax and agreement errors prior to publication.

The article is interesting and introduces novel peptide targets for CPV. However, I recommend a Major Revision. The authors need to adjust the forced interpretation of the ELISpot data, better justify the exclusive use of linear antigens, detail the statistical assumptions, correct the ethics protocol date, and undergo a thorough English grammar review.

 

Comments on the Quality of English Language

• The English language presents flaws and requires mild to moderate improvement. While the text is generally fluid and logically structured for a scientific article, there are grammatical and agreement errors that a native copy-editor would easily catch.
• Examples of flaws in the text:
• At the beginning of the discussion (line 360): "CPVs are increasingly recognized as a clinically relevant pathogens...". Using the singular article "a" with the plural noun "pathogens" is a clear grammatical error.
• On line 369: "For a structural perspective...". The correct phrasing in academic English to start this sentence would be "From a structural perspective...".
• On line 281: "maintained baseline level". Ideally, this should be pluralized to "baseline levels".
• Recommendation to the Editor/Authors: The manuscript's English is good enough for scientific comprehension but requires professional copy-editing (English proofreading) by a native speaker to correct syntax and agreement errors prior to publication.

R: We sincerely thank the reviewer for these helpful comments on the English language quality of the manuscript. The specific examples pointed out by the reviewer have been corrected, including: “a clinically relevant pathogens” to “clinically relevant pathogens”, “For a structural perspective” to “From a structural perspective”, and “baseline level” to “baseline levels”. In addition, the entire manuscript has been carefully proofread and revised to improve grammar, syntax, clarity, and overall readability. We sincerely appreciate the reviewer’s constructive comments and thoughtful suggestions, which have substantially improved the quality and scientific rigor of our manuscript. We hope that the revised version satisfactorily addresses all concerns.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 

The authors have reported on a novel and well-designed next gen vaccine development investigation with a practical primary aim of identification of conserved B-cell epitopes within he L1 proteins of CPV1 and CPV2, the focus of the investigation was to evaluate immunogenic potential and cross reactive potential as vaccine candidates. 

The authors effectively report on antigen-specific induction of interferon gamma in immunized mice. This study reports on the selective epitopes that effectively elicited cross reactive antibodies that recognized both CPV1 and CPV2 L1 proteins. Reported data supports the conclusions that epitope based viral like particle display platforms are useful for multivalent CPV vaccine design. Through this work there is the potential application for vaccine design that will fill a critical gap in protective strategies against CPV in practice.  Study findings align with known details regarding HPV, further supporting the application of this platform and methodology for effective vaccine design. As reported, the identification of conserved epitopes capable of inducing cross reactive antibody responses offers an encouraging foundation for the development of vaccines with broader genotype coverage and host protection. 

Line 35, This study identified conserved cross-reactive B-cell epitopes

 

Author Response

Comments and Suggestions for Authors

The authors have reported on a novel and well-designed next gen vaccine development investigation with a practical primary aim of identification of conserved B-cell epitopes within he L1 proteins of CPV1 and CPV2, the focus of the investigation was to evaluate immunogenic potential and cross reactive potential as vaccine candidates. 

The authors effectively report on antigen-specific induction of interferon gamma in immunized mice. This study reports on the selective epitopes that effectively elicited cross reactive antibodies that recognized both CPV1 and CPV2 L1 proteins. Reported data supports the conclusions that epitope based viral like particle display platforms are useful for multivalent CPV vaccine design. Through this work there is the potential application for vaccine design that will fill a critical gap in protective strategies against CPV in practice.  Study findings align with known details regarding HPV, further supporting the application of this platform and methodology for effective vaccine design. As reported, the identification of conserved epitopes capable of inducing cross reactive antibody responses offers an encouraging foundation for the development of vaccines with broader genotype coverage and host protection. 

Line 35, This study identified conserved cross-reactive B-cell epitopes

R: We sincerely thank the reviewer for the positive and encouraging evaluation of our study. We greatly appreciate the reviewer’s recognition of the novelty of this work, the practical relevance of identifying conserved B-cell epitopes within CPV1 and CPV2 L1 proteins, and the potential value of these findings for future multivalent CPV vaccine design. We are also grateful for the reviewer’s acknowledgment that the identified epitopes elicited cross-reactive antibody responses and that the study provides a useful foundation for broader genotype coverage in CPV vaccine development.

Regarding Line 35, we appreciate the reviewer’s suggestion. The sentence has been revised for improved clarity and precision.

Revised text: “This study identified conserved, cross-reactive B-cell epitopes within the L1 proteins of CPV1 and CPV2.”

We thank the reviewer again for the supportive comments and thoughtful assessment of our work.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all the recommendations and revised the manuscript accordingly, while acknowledging the study’s limitations. I believe the manuscript is suitable for publication in its current form.

Author Response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the originality of identifying conserved cross-reactive B-cell epitopes in CPV1 and CPV2 L1 proteins. We also appreciate the constructive comments regarding methodological details, data presentation, and result interpretation. In response, we have revised the manuscript to improve clarity and rigor. Specifically, we added details on peptide conjugation and immunization procedures, corrected relevant text, and revised the Results and Discussion sections to present the findings more cautiously and avoid overinterpretation.

Reviewer 2 Report

Comments and Suggestions for Authors

 

Reviewer’s Report: Point-by-Point Verification of the Revised Manuscript

As an ad hoc reviewer, I have conducted a detailed verification of the revised manuscript "Identification of Conserved Cross-Reactive B-Cell Epitopes in CPV1 and CPV2 L1 Proteins with Vaccine Potential", comparing the new text against the list of requested corrections from the previous review report.

Below is the point-by-point verification report:

1. Exclusive Focus on Linear Epitopes (Introduction)

• Previous Request: Justify the exclusive choice of linear epitopes, given that conformational epitopes (VLPs) are the basis of protective immunity against papillomaviruses.
• Verification Status: Addressed.
• Analysis: The authors revised the Introduction and Discussion to clarify this point. The manuscript now openly acknowledges that VLP-based vaccines and their conformational epitopes are fundamental. However, they adequately justify that conserved linear epitopes hold value for cross-reactive antigen design, immunodiagnostic development, and incorporation into optimized multi-epitope platforms.

2. Structural Characterization and VLPs (Material and Methods / Discussion)

• Previous Request: Resolve the omission regarding the verification of the self-assembly of purified L1 proteins into Virus-Like Particles (VLPs).
• Verification Status: Addressed.
• Analysis: The manuscript addressed the issue transparently, now treating it as a clear limitation. It was added in the Methods and Discussion that no structural analyses (such as electron microscopy or size-exclusion chromatography) were performed to confirm assembly. The authors explicitly state that the native oligomeric state was not determined and that the recombinant proteins expressed in E. coli should not be described as confirmed VLPs.

3. Inadequate ELISpot Design (Material and Methods) and Overinterpretation (Results)

• Previous Request: Correct the overinterpretation of T-cell data induced by very short peptides (7 amino acids) lacking T-helper epitopes.
• Verification Status: Addressed.
• Analysis: The authors rephrased the tone of the conclusions and considerably reduced the previous claim. The text now expressly states that the low number of IFN-γ secreting cells in the peptide-immunized groups did not show a statistically significant difference compared to the negative control. The ELISpot results are now correctly interpreted as limited and merely as supportive observations of antigen-associated cellular responses. The article also suggests that rigorous evaluations require longer peptide constructs and helper epitopes in the future.

4. Lack of Neutralization Assay (Results / Discussion)

• Previous Request: Moderate the claim of "vaccine potential," since the binding visualized in Western Blot/ELISA does not guarantee neutralization of the actual virus.
• Verification Status: Addressed.
• Analysis: The manuscript was adjusted to reflect this limitation. The authors added that the neutralizing activity and protective efficacy remain undetermined and emphasize that antibody binding detected in vitro does not necessarily indicate neutralizing capacity, requiring future assays with live virus.

5. Over-optimism about Peptides (Discussion)

• Previous Request: Focus on the fact that isolated epitopes must be combined into larger constructs (multi-epitopes) to have potential practical use.
• Verification Status: Addressed.
• Analysis: The Discussion now highlights that minimal linear motifs, such as TPSGSLV and TVVDNTR, elicit weak responses and are "unlikely to function effectively as standalone vaccine antigens". They reinforce that the utility of the peptides lies in their incorporation into carrier formulations, nanoparticle display systems, or multi-epitope antigens.

6. Statistical Analysis (ANOVA Assumptions)

• Previous Request: Report the performance of normality and variance homogeneity tests prior to applying ANOVA.
• Verification Status: Addressed.
• Analysis: Section 2.9 (Statistical Analysis) was updated to indicate that data normality was assessed by the Shapiro-Wilk test, and homogeneity of variances by Levene's test, prior to using parametric analyses.

7. Bioethics Issues: Date Inconsistency

• Previous Request: Correct the date and year of ethical approval (the 2026 number physically linked to March 2024 in the previous text).
• Verification Status: Addressed.
• Analysis: The approval date was corrected in the Institutional Review Board Statement to the number "2026-33, Mar 9, 2026".

8. Bioethics Issues: Animal Welfare

• Previous Request: Include monitoring and humane endpoint criteria due to the use of Freund's Complete Adjuvant (FCA).
• Verification Status: Addressed.
• Analysis: In section 2.6, the authors added information indicating daily monitoring for general health and injection-site reactions. Rigorous humane endpoint criteria were included, such as severe local inflammation, weight loss exceeding 20%, and marked signs of distress.

•  

Final Reviewer's Report and Recommendation: The authors were extremely diligent in responding to the scientific queries, correcting flaws in data interpretation, softening claims about peptide efficacy without neutralization, and clarifying structural limitations, which brought high rigor to the new version of the article.

Recommendation: The manuscript can be Accepted for Publication (Accept), conditioned only on the mandatory correction of minor residual English errors by the copy-editing/proofreading team — specifically on line 406, removing the article "a" before the plural noun "pathogens".

Comments on the Quality of English Language

• •  

   English Language Quality

  • Previous Request: Perform professional copy-editing throughout the manuscript to correct issues such as "recognized as a clinically relevant pathogens", "For a structural perspective", and "maintained baseline level".
  • Verification Status: Partially Addressed.
  • Analysis: It is notable that there were improvements in the overall writing of the text; specific errors were resolved, such as the change to "maintained low baseline levels". However, a thorough proofreading seems not to have been fully completed. For instance, the classic grammatical error of number agreement pointed out in my previous review ("CPVs are increasingly recognized as a clinically relevant pathogens...") still persists on line 406 (Discussion) of this new version.
•  

Author Response

We thank the reviewer for the concise and accurate summary of our study. We appreciate the recognition that our work combines bioinformatics prediction with mouse-based experimental validation to identify conserved cross-reactive linear B-cell epitopes in CPV1 and CPV2 L1 proteins. We have carefully addressed all comments and revised the manuscript to improve clarity, methodological rigor, and overall presentation.

Author Response File: Author Response.pdf