Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors developed and evaluated an ASFV vaccine consisting of a multi-epitope protein antigen and chitosan nanoparticles. Some comments are below:

Please clarify in the title that chitosan-based nanoparticles are used.

Line 48. Is the ASFV vaccine approved only in Vietnam? What about other countries?

Line 73. What is the likelihood of new strains emerging?

Line 86. Protein vaccines are generally highly immunogenic and do not require a delivery system. Therefore, please justify the need for a delivery system and provide references.

Lines 89-91. “The identification and incorporation of important immune response stimulators are key to the design of highly effective vaccines. The immune correlates of protection against ASFV are poorly understood and thus remain an obstacle to vaccine development.” This is unclear. Please rephrase. What are “immune correlates”?

Line 103. Please provide information about the chitosan-based nanoparticles.

Line 106. Why were multi-epitope vaccines chosen?

Line 284. "Using this approach…" Blast alignment does not allow epitope determination, so this conclusion is premature. Please describe the tools that allowed epitope determination.

Line 298. Please provide electrophoresis of the resulting protein.

Lines 305-310. How was the protein encapsulated in chitosan? How was the amount of encapsulated protein determined? Is STING a control here? For comparison, please provide the size and polydispersity index of the empty chitosan particles.

Sections 3.6-3.9. Analysis of the multi-epitope protein without chitosan nanoparticles and unloaded chitosan particles is not provided for comparison. Therefore, the conclusions reached are questionable.

Lines 433-435. "We directly address the challenge posed by ASFV genetic diversity..." This conclusion is questionable. The vaccine's efficacy against different strains was not assessed here.

Line 443. Neutralization tests were not included in this study, so this conclusion is also questionable.

Author Response

We would like to thank the reviewer for their insightful comments.  We have altered the manuscript in response to the coments as described in the attached pdf file. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors focus on the critical need for the prevention and control of African swine fever (ASF) and designed a multiepitope subunit vaccine based on mannose-conjugated chitosan (M-CS) nanoparticles. The safety and immunogenicity of the vaccine were verified through in vitro experiments and animal immunization trials, providing a valuable candidate for vaccine development in this field. Despite the idea is interesting, I still have some concerns regarding the fact:

 

1. In the animal grouping, it is not clear whether the ASF protein in the "ASF protein + M-CS ADU S100" group is encapsulated by nanoparticles. It is recommended to clearly state whether the antigen in the "ASF protein + M-CS ADU S100" group is in a free form in the grouping description, forming a clear contrast with the encapsulated group to facilitate readers' understanding of the logic of the experimental design.
2. Cytokine detection only focused on IL-6 and IL-10, lacking analysis of key antiviral cytokines such as IFN-γ and TNF-α, which cannot fully reflect the Th1/Th2 immune balance. It is suggested to add the detection of cytokines such as IFN-γ, TNF-α, and IL-4 to analyze the bias of the Th1/Th2 immune response.
3. In the flow cytometry detection, detailed information of the antibody panel (such as antibody clone numbers and fluorescent labels) was not provided, and only "Table 3" was mentioned without presenting the content of Table 3, which affects the reproducibility of the experiment.
4. Immunological time points were only set at DPV 22 and DPV 42, lacking long-term monitoring of immune persistence, making it impossible to evaluate the maintenance time of antibody and cellular immunity. It is recommended to add immunological persistence monitoring time points (such as DPV 90 and DPV 180) to detect the maintenance of antibody titer, affinity, and cellular immunity.
5. Some results (such as T cell proliferation and CD8⁺T cell frequency) showed a trend of difference between the vaccine group and the control group, but did not reach statistical significance.
6. The differences in epitope selection, linker design, and immunological effects between the multiepitope design of this study and the reported ASFV multiepitope vaccines (such as the mRNA vaccines and multivalent subunit vaccines mentioned in the paper) were not fully discussed, making it impossible to highlight the advantages of this study. It is suggested to add a discussion section to compare the advantages and disadvantages of this study with the reported ASFV multiepitope vaccines and nanoparticle-delivered vaccines, and emphasize the advantages of this study in epitope conservation, delivery efficiency, and safety.

Author Response

We would like to thank the reviewer for their insightful comments.  We have altered the manuscript in response to the coments as described in the attached pdf file. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript “Vaccination with an African Swine Fever Virus Multiepitope Protein Nanoparticle-based Subunit Vaccine Elicits Robust Immune Responses in vivo” describes the use of in-silico modeling and prediction tools to engineer and then production of a synthetic multiepitope ASF protein containing key immunogenic ASFV sites. The multiepitope ASF protein was expressed in E.coli and entrapped into mannose-conjugated chitosan (M-CS) nano particles for vaccine formulation. The  vaccine was delivered intramuscularly to pigs. T- and B-cell responses were evaluated and showed (although not significant) increase in specific T-rsponses and increase in humoral immune response.

 

Comments:

1. Section 2.2: the authors need to describe more the process of chosing the epitopes and synthesis afterwards; this section is lacking. The sequences were codon optimized for human expression, expressed in E. Coli and then inoculated in pigs. Could not be more helpful if the sequences were codon-optimized for E. Coli or even better for pig expression?
2. Section 3.1: protein expression in E.coli is missing in the this section? Did the authors checked post translation modifications?
3. Section 3.3: the authors mentioned that live weight was measured on D42 using measuring tape; please provide a reference or more details how this was done. Different nomenclatures were used to describe the vaccine and derivatives (e.g M-CS-ASF-NP); it would be helpful to unify this on all sections of the manuscript.
4. 7: I would encourage the authors to show the flow cytometry blots instead/or beside the bars.

Author Response

We would like to thank the reviewer for their insightful comments.  We have altered the manuscript in response to the coments as described in the attached pdf file. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The new additions to the manuscript made a big difference. The quality of the paper had improved, and all my questions were addressed. No more comments.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed all comments. There is now better presentation of the results and other sections.