Oral Immunization with the C488A Live-Attenuated Mutant of Coxsackievirus B4E2 (CVB4E2) Induces Potent Immune Response and Protects Balb/c Mice Against Lethal Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study of the effects of domain V mutations in CVB4 was done to assess the ability to generate attenuated strains for oral vaccines.  It is an interesting study that did find one mutation that reduced the murine virulence of the highly pathogenic CVB4-E2 and generated protective immunity in the murine model.  It is marred by the description of the mutations and sequence of the primers used to generate them.  I note that the authors used the CVB3 E2 (GenBank Accession AF311939) for this study but the numbering does not correspond to the CVB3 E2 sequence given by GenBank.  The primers also have differences from the AF311939 sequence as well as the mutations that they were designed to produce.  Specifically, there are differences in the sequence given in Figure 1A at nt 481,482, 489, and 490 from the AF311939 sequence.  Are these differences present in the CVB3 E2 virus the authors used?  Is there a GenBank accession for this strain that corresponds with these differences? I also note that the G485A primers have a 2 nt (489-490) deletion in comparison to the sequence in Figure 1 and the C473T-R primer has a deletion of the nt that would anneal to nt481.  These may be due to errors in Table I and in Figure 1A but as the authors are publishing data as to attenuating mutations, these errors are critical.  At present, the authors have not provided sequences of the viral genomes derived from these mutated cloned genomes which would verify what the differences are from the parental CVB4 E2 strain.  Indeed, the authors  have not provided a sequence of the parental strain that conforms to Figure 1A.  Therefore, the work cannot be assessed without this data. 

Comments on the Quality of English Language

There are relatively minor English usage errors but the manuscript would be improved by editing by a writer with skills in English. 

For example, paragraph 1, Page 2: CVB4, likely all human enteroviruses is mainly transmitted directly or undirectly among humans by fecale oral route. fecale should be fecal.

Page 4, Section 2.5: "The differents produced mutants, carrying single mutations were engineered using PCR-based site-directed specific mutagenesis method involving two rounds of PCR of the ΔpRib-CVB4E2/T7 DNA." would be better as "The clones carrying single mutations were engineered using PCR-based site-directed specific mutagenesis method involving two rounds of PCR of the ΔpRib-CVB4E2/T7 DNA."

Page 8, 2nd paragraph of Results: "Results of virus mutants titrations by Reed and Muench method revealed 3 Log drop in infectivity for the C486A mutant strain with a viral titer of 1.2 x 103 TCID50." would be better as "Results of titration of virus mutants revealed 3 Log drop in infectivity for the C486A mutant strain with a viral titer of 1.2 x 103 TCID50."

Page 13, 2nd paragraph: "Mice of group G3 receiving just a prime-immunization at day 0 with the live-attenuated strain, then challenged at day 35 with the pathogenic CVB4E2 strain showed a less decreasing in their weight evaluated at 5%." would be better as "Mice of group G3 receiving just a prime-immunization at day 0 with the live-attenuated strain, then challenged at day 35 with the pathogenic CVB4E2 strain showed only a 5% decrease in body weight."

Author Response

Responses to the Reviewer comments:

* "This study of the effects of domain V mutations in CVB4 was done to assess the ability to generate attenuated strains for oral vaccines. It is an interesting study that did find one mutation that reduced the murine virulence of the highly pathogenic CVB4-E2 and generated protective immunity in the murine model. It is marred by the description of the mutations and sequence of the primers used to generate them."

Response: first, we would like to thank very much the reviewer for all her/his efforts in depth reading the entire manuscript. We are truly grateful to the reviewer for all the very pertinent remarks that were made, demonstrating his extensive scientific knowledge on the research topic addressed by our team. His insightful observations remarkably identified errors, particularly in the nucleotide sequences, during the drafting of the manuscript. We would like to express our apologize for these inattentional mistakes…

 

* "I note that the authors used the CVB3 E2 (GenBank Accession AF311939) for this study but the numbering does not correspond to the CVB3 E2 sequence given by GenBank. The primers also have differences from the AF311939 sequence as well as the mutations that they were designed to produce.  Specifically, there are differences in the sequence given in Figure 1A at nt 481,482, 489, and 490 from the AF311939 sequence. Are these differences present in the CVB3 E2 virus the authors used?  Is there a GenBank accession for this strain that corresponds with these differences? I also note that the G485A primers have a 2 nt (489-490) deletion in comparison to the sequence in Figure 1 and the C473T-R primer has a deletion of the nt that would anneal to nt481. These may be due to errors in Table I and in Figure 1A but as the authors are publishing data as to attenuating mutations, these errors are critical." 

Response: Absolutely, we used the CVB4E2 strain for all genomic construction. The sequence of this strain is with the ascension number AF311939. We agree with the reviewer, some sequence errors unintentionally were introduced into the Figure and into the primer sequences by the student who wrote the first draft of article.

We have carefully reviewed our sequences and figure 1A structure and we then corrected all sequence errors both in the figure 1A and in the primer sequences in Table 1 (including the nucleotide numbering and deletion) according to the GenBank sequence of the strain. In addition, as recommended by the reviewer, we added as supplementary Figure the entire sequence of the 5’UTR region including the IRES element and we highlighted the sequence of the domain V of the IRES.

Now, Figure 1A and Tables 1 present new and corrected sequences and new nucleotide numbering (nucleotide positions).

Thank you very much for drawing our attention to this essential aspect of the work!!!

 

* At present, the authors have not provided sequences of the viral genomes derived from these mutated cloned genomes which would verify what the differences are from the parental CVB4 E2 strain.  Indeed, the authors have not provided a sequence of the parental strain that conforms to Figure 1A.  Therefore, the work cannot be assessed without this data." 

Response: We completely agree with the reviewer on this point. Thereby, we added as recommended by the reviewer Figure 3 in supplementary material presenting the entire sequence of the 5’UTR of the parental CVB4E2 strain and we highlighted the sequence of the IRES domain V containing all mutations introduced. Figure 1A is now conforms to the sequence of parental strain.

 

* paragraph 1, Page 2: CVB4, likely all human enteroviruses is mainly transmitted directly or undirectly among humans by fecale oral route. fecale should be fecal.

Response: Sorry for the mistake, it was corrected.

 

* Page 4, Section 2.5: "The differents produced mutants, carrying single mutations were engineered using PCR-based site-directed specific mutagenesis method involving two rounds of PCR of the ΔpRib-CVB4E2/T7 DNA." would be better as "The clones carrying single mutations were engineered using PCR-based site-directed specific mutagenesis method involving two rounds of PCR of the ΔpRib-CVB4E2/T7 DNA."

Response: Thanks for the correction of the sentence. We corrected it as suggested by the reviewer.

 

* Page 8, 2nd paragraph of Results: "Results of virus mutants titrations by Reed and Muench method revealed 3 Log drop in infectivity for the C486A mutant strain with a viral titer of 1.2 x 103 TCID50." would be better as "Results of titration of virus mutants revealed 3 Log drop in infectivity for the C486A mutant strain with a viral titer of 1.2 x 103 TCID50."

Response: Thanks for the correction of the sentence. We corrected it as suggested by the reviewer.

 

* Page 13, 2nd paragraph: "Mice of group G3 receiving just a prime-immunization at day 0 with the live-attenuated strain, then challenged at day 35 with the pathogenic CVB4E2 strain showed a less decreasing in their weight evaluated at 5%." would be better as "Mice of group G3 receiving just a prime-immunization at day 0 with the live-attenuated strain, then challenged at day 35 with the pathogenic CVB4E2 strain showed only a 5% decrease in body weight."

Response: Thanks for the correction of the sentence. We corrected it as suggested by the reviewer.

Reviewer 2 Report

Comments and Suggestions for Authors

As the authors summarize in the conclusion, this work focused on the IRES of Coxsackievirus B4 and demonstrated that the designed attenuated C486A mutant effectively induces an immune response and protects Balb/c mice from lethal challenge, showing its potential as a promising vaccine candidate. The tests include in vitro transfection, translation, neutralization antibody titers via mouse challenge, and cytokine quantification. No ethical issues or inconsistencies in results were observed. The value of this paper lies in its technical aspects, with little description of novelty. This reviewer recommends publication in Viruses after the following minor to major revisions.

 

The figures need refinement. The graphical presentation is poor relative to what is described in the Methods section and Figure Legends, diminishing the value of the manuscript.

 

1. There are many bar graphs. The figure legends mention error bars and statistical test results, but no trace of these is visible in the figures. Are the error bars too small to recognize? If comparisons are made using error bars and multiple measurements, appropriate statistical tests should be performed, and at minimum, numerical tables should be provided if the graphs are not clearly interpretable.
2. Typos are present in the vertical axis labels of some graphs. For example, "liter" appears sometimes lowercase, sometimes uppercase. The text has the same issue, so please standardize. In Fig. 2C, "pgg/mL" has an extra "g".
3. For Fig. 3, are comparisons between groups performed? The legend suggests so. Also, distinguishing G1-G6 in Fig. 3B and C is extremely difficult. Please improve the presentation. Without numerical tables, the value of the plots is significantly compromised.
4. Confirmation of mutant construction is central to this study; if sequencing data exists, please include it in the supplement.

Author Response

Responses to the Reviewer comments:

* As the authors summarize in the conclusion, this work focused on the IRES of Coxsackievirus B4 and demonstrated that the designed attenuated C486A mutant effectively induces an immune response and protects Balb/c mice from lethal challenge, showing its potential as a promising vaccine candidate. The tests include in vitro transfection, translation, neutralization antibody titers via mouse challenge, and cytokine quantification. No ethical issues or inconsistencies in results were observed. The value of this paper lies in its technical aspects, with little description of novelty. This reviewer recommends publication in Viruses after the following minor to major revisions.

Response: We would like to thank very much the reviewer for all her/his efforts in depth reading the entire manuscript. We are truly grateful to him for all the very pertinent remarks that were made, demonstrating his extensive scientific knowledge on the research topic addressed by our team.

 

* There are many bar graphs. The figure legends mention error bars and statistical test results, but no trace of these is visible in the figures. Are the error bars too small to recognize? If comparisons are made using error bars and multiple measurements, appropriate statistical tests should be performed, and at minimum, numerical tables should be provided if the graphs are not clearly interpretable.

Response: Thanks very much for the important comment. We now improved the quality of all Figures and we incorporated error bars relative to the statistical test of results

 

* Typos are present in the vertical axis labels of some graphs. For example, "liter" appears sometimes lowercase, sometimes uppercase. The text has the same issue, so please standardize. In Fig. 2C, "pgg/mL" has an extra "g".

Response: Thank you for bringing these errors to our attention. We corrected them in text and Figures.

 

* For Fig. 3, are comparisons between groups performed? The legend suggests so. Also, distinguishing G1-G6 in Fig. 3B and C is extremely difficult. Please improve the presentation. Without numerical tables, the value of the plots is significantly compromised.

Response: Thanks for the pertinent comments. Unfortunately, in Figure 3C plots related to some mice groups (G1, G2, G4 and G6) are compromised because we didn’t reveal any mortality among these mice groups and the percentage of survival mice remains the same as 100%. Only G3 and G5 revealed a decrease percentage in survival mice.

 

* Confirmation of mutant construction is central to this study; if sequencing data exists, please include it in the supplement.

Response: As recommended by the reviewer, we added in the supplementary material a Figure 3 reporting the 5’UTR sequence of the parental strain with the position of introduced mutations in domain V of the IRES.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have resolved the issues that I raised in the first version of this manuscript.

Comments on the Quality of English Language

Minor issue: there are still typographic and grammar errors in the text.  I have noted those that I found below.

Throughout the text both versions of the words immunization/immunisation, immunized/immunised are used. This is also true for analyzed/analysed. Only one version should be used throughout the text.

The word efficiency is misspelled in the abstract, twice in the first paragraph of page 3, once in the second paragraph of page 10 and in the first and second paragraph of the Discussion (Section 4). The word immunogenicity is misspelled in the first paragraph of page 3 and in the second paragraph of the discussion. The word nucleotide is misspelled in section 2.5 and section 3.1, in the legend for Figure 1. Invitrogen is misspelled in section 2.5; different is misspelled in section 2.5; transcription is misspelled in section 2.6; finally is misspelled in section 2.7; facility is euthanized and homogenized are misspelled in section 2.12; supplementary is misspelled in the second paragraph of section 3.1; parental is misspelled in the third paragraph of section 3.1; introduction is misspelled in the third and fourth paragraphs of section 3.1; mutation is misspelled in the first paragraph of page 10; negative is misspelled in the first paragraph of page 14; translation is misspelled in the first paragraph of page 16.

In the first paragraph of the Introduction page 2, the sentence “CVB4, likely all human enteroviruses is mainly transmitted directly or undirectly among humans by fecal-oral route.” should be “CVB4, like all human enteroviruses, is mainly transmitted directly or indirectly among humans by fecal-oral route.”

The sentence “Group 1 (G1) Balb/c mice received at day 0 a prime orally immunization with a dose of 105 TCID50 of the live-attenuated strain.” should be “Group 1 (G1) Balb/c mice received at day 0 an oral prime immunization with a dose of 10⁵ TCID₅₀ of the live-attenuated strain.” And the sentence “Group 5 (G5) mice were used as positive control, mice of this group were administered orally 10⁵ TCID₅₀ of CVB4E2 pathogenic strain at day 0.”

The sentence “HeLa cells was used in this study to experimentely evaluated the replicative capacities of generated mutants comparing to the parental CVB4E2 pathogenic strain.” should be “HeLa cells were used in this study to experimentally evaluate the replicative capacities of generated mutants comparing to the parental CVB4E2 pathogenic strain.”

i.e. is i,e in the third paragraph of section 3.1.

In the sentence “The mode of the natural contamination of CVB4 strains is mainly the fecal-oral route (digestive tract), then we have choosed in the present study the oral route for the mice immunisations and challenges”, choosed should be chosen.

 

 

Author Response

Responses to the Reviewer #1 comments:

* "The authors have resolved the issues that I raised in the first version of this manuscript."

Response: Thanks very much to the reviewer for all efforts and contribution to improve the quality of the manuscript.

* "Minor issues: there are still typographic and grammar errors in the text":  

Response: Misspellings were carefully detected by the reviewer in the text. We are grateful for the revision of the text conducted by the reviewer. All errors were corrected in the new version of the manuscript and highlighted.

Reviewer 2 Report

Comments and Suggestions for Authors

Journal: Viruses, Manuscript ID: viruses-4119802

Type of manuscript: Article

Title: Oral immunization with the C486A live-attenuated mutant of coxsackievirus B4E2 (CVB4E2) induces potent immune response and protects Balb/c mice against lethal infection. 

Authors: Jawhar Gharbi *, .... , Manel Ben M’hadheb

Human Virology and Viral Diseases

 

As pointed out previously, the value of this paper lies in its technical aspects, and not much is written regarding its novelty. Therefore, the materials used and the analysis of the results are the core of this paper.

 

Regarding previous comment 4, the reviewer is not simply asking to show the mutation sites, but is expecting data demonstrating that "the mutant virus created is indeed the virus with the designed mutations." Since this is something that should have been confirmed during the research process, new experiments are not expected; however, it is the starting point for showing the integrity of the materials used. This reviewer trusts that the authors conducted subsequent experiments after performing specific verifications.

 

Previous comments 1-3 concerned the presentation of the analysis results, and the "distribution" within the data in the bar graphs has been improved.

Author Response

Responses to the Reviewer #2 comments:

* “As pointed out previously, the value of this paper lies in its technical aspects, and not much is written regarding its novelty. Therefore, the materials used and the analysis of the results are the core of this paper.

Regarding previous comment 4, the reviewer is not simply asking to show the mutation sites but is expecting data demonstrating that "the mutant virus created is indeed the virus with the designed mutations." Since this is something that should have been confirmed during the research process, new experiments are not expected; however, it is the starting point for showing the integrity of the materials used. This reviewer trusts that the authors conducted subsequent experiments after performing specific verifications.”

Response: We would like to thank very much the reviewer for reading in depth of our manuscript and for his/her interest in the work.

We understand the reviewer’s concern about the integrity of the initial material, and we inform him/her that all genomic constructs have undergone systematic genomic identity checks through systematic sequencing of amplification PCR products since the production of mutant clones pcDNA-E2, then the results of PCR site-directed mutagenesis products, and finally cell transfection and the production of viral mutants.

Results of systematic sequencing of the part of CVB4E2 IRES conducted in each step of molecular experimentation using the automatic DNA sequencing revealed the presence of the different mutations introduced in the appropriate positions. No supplementary or deletion mutations were revealed.

In addition, we added as reference in Figures 3 in supplementary material the entire sequence of IRES of the parental construct pcDNA-E2.

 

* “Previous comments 1-3 concerned the presentation of the analysis results, and the "distribution" within the data in the bar graphs has been improved.”

Response: Thanks for the comments.