Utilizing Immunoinformatics for mRNA Vaccine Design against Influenza D Virus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The author described computational designs of mRNA vaccine candidates for Influenza D virus. The topic is potentially interesting and important given the risks of zoonotic spillover of IDV from animals to humans. A comprehensive computational tools were used for T and B cell epitope prediction and antigenicity prediction of designed mRNAs. However, experimental data testing these mRNAs either in vitro or in vivo are lacking. 

Comments on the Quality of English Language

Writing is mostly clear and easy to follow. Very minor edits may be required.

Author Response

REVIEWER 1:

Comment 1: The author described computational designs of mRNA vaccine candidates for the Influenza D virus. The topic is potentially interesting and important given the risks of zoonotic spillover of IDV from animals to humans. Comprehensive computational tools were used for T and B cell epitope prediction and antigenicity prediction of designed mRNAs. However, experimental data testing these mRNAs either in vitro or in vivo are lacking. 

Response: The stage of the work is the computational design of the vaccine construct in which this will make up for the next step including the trails in various animal models depending on the availability of funding. This stage is self funding.

 

Comment 2: The writing is mostly clear and easy to follow. Very minor edits may be required.

Response: Accepted and reflected.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have developed an mRNA vaccine construct against Infectious Delta Virus (IDV) using immunoinformatic methods, studying antigenic Hemagglutinin Esterase Fusion (HEF) sequences from IDV isolates. Comprehensive epitope prediction and selection resulted in a vaccine prototype comprising nine epitopes, an adjuvant, and MHC I-targeting domain.The vaccine exhibits antigenicity, non-allergenicity, and non-toxicity with promising physicochemical properties and stable structural characteristics. Molecular docking simulations implies strong binding affinity with toll-like receptors, indicating potential efficacy. 

 

Here are my comments:

1.Fig 3:

In the text the authors write:

“After conducting several analyses, eleven (11) epitopes comprising 3 B-cell epitopes, 4 CTL

epitopes, and 4 HTL epitopes were selected for the primary vaccine construct. Additional components, such as the 5' UTR, Kozak sequence, tPA, etc. were incorporated into the primary construct forming the mRNA vaccine, as illustrated in Figure 3 below.”

However, in Fig 3, the schematic diagram shows 3 HTL, 4 B cell and 4 CTL epitopes- Please clarify.

 

2.In the text section of “Prediction of Physicochemical Properties of the Vaccine Candidate”- it contains 101 negatively charged (Asp + Glu)- This should be Asp-Glu instead of negatively charged (Asp + Glu). This should be clarified in the table as well.

 

3. Proteins with negative GRAVY values is hydrophilic and positive GRAVY values is hydrophobic. However, in the results section the authors mention the GRAVY value is negative at 0.058 but is hydrophobic. Please go through the results section and discussion section as there is discrepancy between hydrophobicity and hydrophilicity. Please clarify whether the GRAVY score is negative and hydrophilic. Change accordingly throughout the text and table.

 

4. In the discussion section the authors mention” The vaccine construct's stabilized structure

comprises 47.35% alpha-helix, 14.27% extended strands, and 34.25% random coils, as confirmed by the analysis.  However, table 6 shows that the alpha helix is 37.65%, extended strand is 19.01% and the random coils is 34.25%- Please clarify.

 

5. Has the vaccine construct being tested invitro?

 

Author Response

Here are my comments:

1. Fig 3: In the text, the authors write:

“After conducting several analyses, eleven (11) epitopes comprising 3 B-cell epitopes, 4 CTL

epitopes, and 4 HTL epitopes were selected for the primary vaccine construct. Additional components, such as the 5' UTR, Kozak sequence, tPA, etc. were incorporated into the primary construct forming the mRNA vaccine, as illustrated in Figure 3 below.”

However, in Fig 3, the schematic diagram shows 3 HTL, 4 B cells, and 4 CTL epitopes- Please clarify.

Response: Accepted. It has been clarified.

 

2. In the text section of “Prediction of Physicochemical Properties of the Vaccine Candidate”- it contains 101 negatively charged (Asp + Glu)- This should be Asp-Glu instead of negatively charged (Asp + Glu). This should be clarified in the table as well.

Response: Clarified. The positive sign meant Asp “and” Glu, it does not refer to the charge.

 

3. Proteins with negative GRAVY values are hydrophilic and positive GRAVY values is hydrophobic. However, in the results section, the authors mention the GRAVY value is negative at 0.058 but is hydrophobic. Please go through the results section and discussion section as there is a discrepancy between hydrophobicity and hydrophilicity. Please clarify whether the GRAVY score is negative and hydrophilic. Change accordingly throughout the text and table.

Response: Accepted. Changes have been made.

 

4. In the discussion section the authors mention” The vaccine construct's stabilized structure comprises 47.35% alpha-helix, 14.27% extended strands, and 34.25% random coils, as confirmed by the analysis.  However, table 6 shows that the alpha helix is 37.65%, the extended strand is 19.01% and the random coils are 34.25%- Please clarify.

Response: Accepted. Changes have been made.

 

5. Has the vaccine construct been tested in vitro?

Response: No. The stage of the work is the computational design of the vaccine construct in which this will make up for the next step including the trails in various animal models depending on the availability of funding. This stage is self funding.

Reviewer 3 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Author Response

Comments: Please rephrase the sentence.

35. Page no 15: The stability of the vaccine construct is found to be 35.40, indicating that it is stable.

Response: Accepted and reflected.

 

79. Page No 15: The Authors have stated that the aliphatic index of the vaccine construct is 79.04, suggesting its potential thermostability. It would be amicable if authors could write threshold values in Table 5.

Response: Accepted. The threshold has been included. The higher the aliphatic index, the more stable a protein is. Therefore, a threshold of > 50 was set.

 

• What PDB ID were used in the study, the manuscript doesn’t mention anything about that.

Response: Not applicable

 

1. Page no 19 and Page 24: The energy values for the corresponding Docking Scores are missing.

Response: The HDock server was used for the docking. The e-values of the docking scores were not predicted by the server.

 

1. In the context of Cytotoxicity, why didn’t the authors perform any Cell Viability Assay?

Response: The authors do not have the funds to perform this task, it has been penciled down for the next stage of the preclinical evaluation of the vaccine construct.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I still think the scope and significance of this manuscript is limited by the lack of any experimental data to support the computational design. However, given the fact that this was submitted to BioMedInformatics, I guess it does fit into the scope of this journal.

Comments on the Quality of English Language

Writing reads well. 

Author Response

Dear Reviewer, thank you for your valuable feedback. We understand your concern regarding the lack of experimental data to support the computational design presented in our manuscript. Unfortunately, we are currently unable to perform the experimental validation due to budget constraints, as our research is self-funded and we do not have the necessary financial resources at this time.

We appreciate your understanding and hope that the significance and potential impact of our computational findings will still contribute meaningfully to the field within the scope of BioMedInformatics.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments and made the changes to the manuscript accordingly.

Author Response

Dear Reviewer, thank you for your thorough review and constructive comments. We are pleased to hear that our revisions have adequately addressed your concerns and improved the manuscript. We appreciate your feedback and support throughout this process.

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations!!!

Author Response

Dear Reviewer, thank you very much for your kind words and congratulations. We appreciate your positive feedback and are delighted that our work has met your expectations.