Adenovirus-Mediated Expression of Dengue Virus 2 Envelope Ferritin Nanoparticles Induced Virus-Specific Immune Responses in BALB/c Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Tennakoon et al. described their approach for viral vector based dengue virus vaccine. The authors showed successful production of the adenovirus, and the protection of the vaccine in mice. However, some questions need to be addressed:

1.  Line 40-50, please add relevant literatures in the case of dengue vaccine.

2. Section 2.1 is missing the strain information of DENV-2.

3. Can the authors elaborate further about why they chose DENV-2 but not the other 3 serotypes?

4. Section 2.9, is there a specific reason for only using female mice?

5. In Figure 2, the study design is missing a negative control of using the empty plasmid.

6. In Figure 1-3, it is a bit concerning that the anti-Myc cannot detect the E-H. The author explained that the segment is in the stacking gel region but this is not convincing. The usage of stacking gel is to line up all the protein samples loaded on the gel, so that they can enter the resolving gel at the same time. With the bands of the marker nicely distributed around, it is unlikely that specific region is in the stacking gel.

7. In Figure 4, can author measure the neutralizing titer in the blood sample? Also, can the authors test if this vaccine has any cross activity with DENV-1, DENV-3 or DENV-4?

Author Response

Responses to Reviewer Report: Reviewer: 1

1. Line 40-50, please add relevant literatures in the case of Dengue vaccine.

              Response- relevant literatures were cited as requested

2. Section 2.1 is missing the strain information of DENV-2.

                Response- Strain information was added (line 79)

3. Can the authors elaborate further about why they chose DENV-2 but not the other 3 serotypes?

Response- according to the previous references DENV2 serotype is the most prevalent dengue virus in the world

(https://doi.org/10.1016/j.meegid.2020.104391)

(10.1099/acmi.0.000567.v4)

Therefore, we used the DENV2 virus to elucidate the preliminary ability of the ferritin-conjugated dengue envelope to induce an immune response through gene delivery using adenoviruses.

Moreover, currently, we have only dengue 2 virus strain in the lab. In this study, we isolated mRNA for the dengue envelope gene from the wild-type virus (not synthesized). The synthesis of total genes can be a significant cost for preliminary studies.

4. Section 2.9, is there a specific reason for only using female mice?

Response- There were no considerable reasons for selecting female BALB/c mice. However, they showed greater stability in nesting than in male mice.

Furthermore, previous studies have reported that female mice produce potent antibody responses.

(https://doi.org/10.1073/pnas.1805268115)

5. In Figure 2, the study design is missing a negative control of using the empty plasmid.

Response- Empty plasmids were transfected into 293A cells and the image was taken and represented in Figure 2, as requested, and relevant descriptions were included in the main body.

6. In Figure 1-3, it is a bit concerning that the anti-Myc cannot detect the E-H. The author explained that the segment is in the stacking gel region, but this is not convincing. The usage of stacking gel is to line up all the protein samples loaded on the gel, so that they can enter the resolving gel at the same time. With the bands of the marker nicely distributed around, it is unlikely that specific region is in the stacking gel.

Response- Stacking gel related information were removed from the manuscript and detection of two different constructs with Myc and human ferritin heavy chain antibody was indicated. (line 220, 222 and 256,257)

7. In Figure 4, can author measure the neutralizing titer in the blood sample? Also, can the authors test if this vaccine has any cross activity with DENV-1, DENV-3 or DENV-4?

We attempted to obtain plaques from the available DENV2 using different methods several times, which was not successful. Therefore, we failed to perform the PRNT assay as our strains didn’t produce CPE. Moreover, currently, we have only dengue 2 virus strain in the lab. Therefore, it was not possible to perform cross-reactivity experiments using sera.

Our main aim was to demonstrate the functionality of ferritin nanoparticles generated from Adenovirus-mediated delivery and to communicate the technical hints for the development of a robust tetravalent vaccine for dengue or vaccines against other viral strains. Therefore, we used only one serotype of dengue viruses.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. What is the main question addressed by the research?

In the present study, Tennakoon et al described the efficiency of the adenovirus-based vaccine against Dengue virus. The authors tried to find if a viral vector vaccine, using genetic material encoded by Dengue envelope and ferritin nanoparticles, effectively stimulated an immune response against the Dengue virus in a murine model. The authors found that the recombinant adenovirus containing Dengue envelope protein 2 along with Ferritin indues higher IgG as compared to the Enveloped virus alone.

2. What parts do you consider original or relevant for the field? What
specific gap in the field does the paper address?

The study provides a comparative analysis of immune responses elicited by adenoviruses expressing the Dengue virus envelope protein alone versus those expressing both the Dengue envelope protein and envelope-human ferritin. This direct comparison helps to evaluate the added benefit of incorporating ferritin nanoparticles into the vaccine design.

By demonstrating the effectiveness of using adenoviral vectors to deliver ferritin nanoparticles, this study proposes a vaccine platform that could be adapted for other pathogens. This could represent a significant advancement in vaccine technology by combining viral vector and nanoparticle-based approaches. Although the finding contributes some knowledge to the field of ferritin-based adenovirus vaccine approach, but this type of study has been done earlier with several other groups in the past both in the case of Dengue virus and other viruses.

3. What does it add to the subject area compared with other published
material?

While there is existing research on viral vector vaccines and ferritin nanoparticles separately and in combination, this study explores the use of ferritin nanoparticles as a carrier within a viral vector system. This approach could enhance the delivery and presentation of the Dengue envelope antigen, potentially improving the immunogenicity of the vaccine. The study shows that the envelope-ferritin nanoparticle vaccine induces a more significant IgG immune response compared to the envelope-only vaccine suggesting that incorporating ferritin nanoparticles can enhance the immunogenicity of viral-based vector vaccines. The observation of the persistence of the immune response in the envelope+ ferritin nanoparticle group adds valuable preliminary data on the duration of immunity provided by this vaccine strategy. This aspect is crucial for knowing long-term vaccine efficacy.

4. What specific improvements should the authors consider regarding the methodology? What further controls should be considered?

·       Cellular Immune Response: In addition to measuring IgG titers, evaluate T cell responses (e.g., IFN-γ production, cytotoxic T lymphocyte activity) to provide a more detailed understanding of this approach.

·       IgG Subtype: Include assay to determine the IgG subtypes (e.g., IgG1, IgG2a) to understand the nature of the immune response elicited by the vaccine (TH1/TH2).

·       Vehicle Control: Use a control group that receives the adenoviral vector without any genetic insert (i.e., an empty adenovirus). This helps distinguish between the effects of the viral vector and the specific antigens being expressed.

·       Negative Controls for Ferritin Nanoparticles: Test a group of mice with ferritin nanoparticles alone (without the viral vector) to determine if ferritin nanoparticles alone induce any immune responses.

·       Cross-Reactivity Control: Test for cross-reactivity by including an additional group that receives a vaccine against a different pathogen. This ensures that the immune responses observed are specific to Dengue virus and not due to non-specific activation.

·       Challenge Studies: After immunization, challenge a subset of the immunized mice with live Dengue virus to directly assess the vaccine’s protective efficacy against infection.

5. Please describe how the conclusions are or are not consistent with the evidence and arguments presented. Please also indicate if all main questions posed were addressed and by which specific experiments.

·       The study concludes that the viral vector vaccine using ferritin nanoparticles is effective in inducing a strong immune response against Dengue virus. This conclusion is supported by the data showing that mice immunized with the envelope-ferritin adenovirus exhibited higher virus-specific IgG titers compared to those immunized with the envelope-only adenovirus. The conclusion is consistent with the evidence presented, as the observed increase in IgG titers directly supports the effectiveness of the vaccine with ferritin nanoparticles.

·       The study suggests that the envelope-ferritin nanoparticle vaccine induces a more robust and persistent immune response than the envelope-only vaccine. The study compares IgG titers between the two groups and reports a more significant immune response in the envelope-ferritin group. This conclusion is consistent with the in vivo data, which shows a prolonged immune response in the envelope-ferritin group, indicating the advantage of using ferritin nanoparticles. While the conclusion is consistent with the evidence, the study would benefit from longer-term follow-up data to support claims of long-term immunity more definitively.

 6. Are the references appropriate?

The references used in this manuscript lack a few of the important references (including ferritin-related studies).

7. Please include any additional comments on the tables and figures and the quality of the data.

Major Comments-

· No IgG subtyping is performed. It could be easily performed using the same sample used for whole IgG titer determination. By comparing the IgG subtype profiles between the envelope-ferritin nanoparticle, envelope, and ferritin, the authors could gain a better understanding of how the ferritin nanoparticles influence the immune response.

·       The authors have not discussed cross-reactivity.  Did they check the immune response against other, non-related antigens to ensure that the observed immune response is specific to the Dengue virus and not due to cross-reactivity with other proteins?

·       No challenge study was performed. After immunization, challenge the mice with a live dengue virus to assess the protective efficacy of the vaccine. This would provide direct evidence of the vaccine’s ability to prevent infection. Also, discuss if any change in the clinical score of animals was observed. What was the effect on the survivability of animals?

Minor comments-

·       Fig 2 (b)- What is the size of protein bands? Add the size of the marker also.

·       The authors mentioned in the abstract that envelope ferritin-immunized mice showed a significant immune response. What type of immune response? Pleases discuss.

·       Provide a reference in line 39.

·       What is PEI in line 119? Pleases explain.

·       What was the concentration of dengue virus used for coating? Why intact dengue virus was used for coating and not E2 protein? What were the positive and negative controls?

·       In line 188, the HRP-conjugated anti-mouse secondary antibody? Which subtype?

·       Please make a separate table for the primer sequence used in this study.

·       Why authors have selected dengue 2 envelope protein and not Dengue 1, 3, 4. Please describe.

·       In fig 4: In (a) what was done with the blood please show in the figure. It looks incomplete figure. What was the concentration of virus used for immunization? Route? (b) this figure is not needed here. The authors have not checked viral presence inside tissues (c) What is this absorbance for? Indicate in Fig (IgG). Use one-way ANOVA to compare the significant differences between all the groups.

Comments on the Quality of English Language

Minor editing is needed.

Author Response

Responses to Reviewer Report: Reviewer: 2

Major Comments-

• No IgG subtyping is performed. It could be easily performed using the same sample used for whole IgG titer determination. By comparing the IgG subtype profiles between the envelope-ferritin nanoparticle, envelope, and ferritin, the authors could gain a better understanding of how the ferritin nanoparticles influence the immune response.

We appreciate the valuable comments of the reviewer, and it is important to advance our study in the future. In this study, we focused only on Adenovirus generation, purification strategy, revealing vaccine strategy by elucidation of primary IgG response only. We are going to improve our study using dengue virus Domain III protein as the primary antigen, and we are supposed to perform IgG subtyping in our future studies.

• The authors have not discussed cross-reactivity.  Did they check the immune response against other, non-related antigens to ensure that the observed immune response is specific to the Dengue virus and not due to cross-reactivity with other proteins?

Response- Thank you for this comment. For Dengue vaccine development project, this would be our first step to proceed. We are agreeing reviewer’s comments but still quite early stage of our long run project. Please accept our results as our first beginning of project. As reviewer criticized, we still needing cross reaction as Flaviviruses do so, and we going to include with other sets of studies. We confirmed that the PBS group did not elicit any immune response against DENV-2, and envelope adenovirus induced no immune responses or it was significantly lower against DENV-2 compared to the envelope ferritin adenovirus immunized. To determine whether the ferritin heavy chain induced a specific immune response, we performed an ELISA using purified ferritin as the coating antigen. The ferritin-specific IgG response was very low and not significant between the groups. Therefore, the envelope ferritin-immunized group induced a significant IgG response against dengue virus only and not against other antigens. The ferritin-specific ELISA data is indicated at the bottom of the document.

 **Upon your request we can include ferritin heavy chain expression data and ferritin specific IgG response data in the manuscript. **

• No Challenge study was performed. After immunization, challenge the mice with a live dengue virus to assess the protective efficacy of the vaccine. This would provide direct evidence of the vaccine’s ability to prevent infection. Also, discuss if any change in the clinical score of animals was observed. What was the effect on the survivability of animals?

Thank you for your suggestion. As previously explained, this is a preliminary study to show the immune response of dengue ferritin nanoparticles expressed by respective recombinant adenoviruses; therefore, we focused mainly on adenovirus generation, purification, and primary immune response. We will apply advanced techniques in further studies in the future.

Minor comments-

• Fig 2 (b)- What is the size of protein bands? Add the size of the marker also.

Response - We apologize for the mistake. We included the size marker right next to the gel image.

• The authors mentioned in the abstract that envelope ferritin-immunized mice showed a significant immune response. What type of immune response? Pleases discuss.

 Response – We have replaced the immune response with IgG response and dengue virus-specific IgG response in the abstract (lines 21 and 22).

• Provide a reference in line 39.

Response - Requested reference was provided as required and additional references were also provided for ferritin nanoparticles (lines 56-65).

• What is PEI in line 119? Please explain.

Response – PEI stands for polyethylenimine. It was mentioned in the manuscript as requested (line 126)

• What was the concentration of dengue virus used for coating? Why intact dengue virus was used for coating and not E2 protein? What were the positive and negative controls?

Response-

We used 100 times diluted (10^-2) DENV2 virus as the coating antigen. We have mentioned this in the revised manuscript (line 189)

The major limitation was that we could not obtain a purified envelope protein of dengue virus with good purity as the coating antigen. Therefore, we used Dengue virus as the coating antigen for ELISA.

Furthermore, we speculated that ferritin nanoparticles mimic the viral structure (round-shaped morphology representing the antigens outside of the cage). Therefore, it can induce a similar immune response to the virus. If the serum contains more virus-specific IgG, virus neutralization activity will also be significant. Therefore, we believe that it is reasonable to evaluate virus-specific IgG response.

• In line 188, the HRP-conjugated anti-mouse secondary antibody? Which subtype?

Response- Thank you for your comment. We changed it to IgG and it was highlighted in the manuscript (line 195).

• Please make a separate table for the primer sequence used in this study.

Response – We have included all the primers in a table, and primer names were included in the manuscript for cloning. The primer names have been highlighted in the manuscript (line 122-123).

• Why authors have selected dengue 2 envelope protein and not Dengue 1, 3, 4. Please describe.

According to the reports, the most prevalent dengue serotype in the world is DENV2. Therefore, we used DENV2 serotypes envelope protein to reveal the primary vaccine strategy.

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10323805/)

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199925/)

Moreover, currently, we have only dengue 2 virus strain in the lab. In this study, we isolated mRNA for the dengue envelope gene from the wild-type virus (not synthesized). The synthesis of total genes can be a significant cost for preliminary studies, if they fail. Our main aim was to demonstrate the functionality of ferritin nanoparticles generated from adenovirus-mediated delivery and to communicate the technical hints for the development of a robust tetravalent vaccine for dengue or vaccines against other viral strains. Here, we elucidated that viral vectors can be used to deliver the genetic code of ferritin nanoparticles, subsequently inducing an IgG response.

• In fig 4: In (a) what was done with the blood please show in the figure. It looks incomplete figure. What was the concentration of virus used for immunization? Route?

Response- We performed the ELISA using the mouse serum isolated from blood and it was indicated using a new figure. The virus concentration and immunization route both are indicated in the figure legend as well as in the manuscript.

 (b) this figure is not needed here.

  Response- This figure was removed from the manuscript as requested.

The authors have not checked viral presence inside tissues

Thank you for the reviewer’s comment. We confirmed the invitro transduction efficiency of the recombinant adenoviruses. And we confirmed specific IgG response which was induced by recombinant adenoviruses. So, we hope it will not greatly affect the conclusion of the immune response.

(c) What is this absorbance for? Indicate in Fig (IgG). Use one-way ANOVA to compare the significant differences between all the groups.

Response- We apologize for the unclear indication. It has been replaced with the optical density in the figure.

Instead of using ANOVA, we simplified the data by using three graphs. The first graph indicates the immune response at week 1. The second graph indicates the immune response at week 4. We compared the immune response of the E-H group at two different time points in another graph, since only E-H induced a significant immune response. Statistical significance was analyzed using the t-test.

Addressing specific comments

4. What specific improvements should the authors consider regarding the methodology? What further controls should be considered?

• Vehicle Control: Use a control group that receives the adenoviral vector without any genetic insert (i.e., an empty adenovirus). This helps distinguish between the effects of the viral vector and the specific antigens being expressed.

Response- Appreciate your valuable comment. This can be explained as follows: the viral vector-only group was used to show whether the vector can induce a specific immune response. We used envelope adenoviruses as the envelope-only control and contained a viral vector as well as the envelope gene. However, the IgG response from the envelope adenovirus was not significant compared to PBS, and envelope ferritin adenoviruses induced a significant IgG response. Therefore, we believe that if we used a vector only, it would not induce a significant immune response similar to that of envelope-only adenoviruses.

• Negative Controls for Ferritin Nanoparticles: Test a group of mice with ferritin nanoparticles alone (without the viral vector) to determine if ferritin nanoparticles alone induce any immune responses.

Resopone - We have immunized mice with human ferritin for another experiment. Those results showed significant human ferritin-specific IgG response in mice but not other antigen-specific IgG responses.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors' efforts to revise the manuscript, and I believe it has improved significantly since the last version. With some minor editions, I recommend this paper to be published in Microbiology Research.

Some concerns to be addressed:

1. In Figure 2a, please specify in figure legend what is used as control. Similarly, in Figure 2b, please explain what is "mock". Is it mock-transcribed with PBS, or empty plasmid?

Also, my last comment about this Figure missing negative control is referring to the whole figure and experiment set-up. Not just Figure 2a. Can authors add those if feasible?

2. In Figure 4, I understand that the DENV-2 strain does not produce plaque. Is it possible to conduct non-CPE based neutralization assay such as FRNT?

Binding result by itself can be less scientifically sound.

Author Response

Responses to Reviewer Report: Reviewer: 1

1. In Figure 2a, please specify in figure legend what is used as control. Similarly, in Figure 2b, please explain what is "mock". Is it mock-transcribed with PBS, or empty plasmid?

Response – Thanks to the reviewer for indicating missed information. In Fig. 2a, we indicated the control as vector transfection control (line 246 highlighted). In the figure legend, we indicated that mock is vector transfection control in line 247 (highlighted).

Also, my last comment about this Figure missing negative control is referring to the whole figure and experiment set-up. Not just Figure 2a. Can authors add those if feasible?

Response - We understand reviewer speculation from this comment. We used negative controls in Fig. 2a and 2b to show that transfection of vector only cannot produce any adenoviruses. Even PCR results, we cannot identify bands in the vector only control revealing the absence of specific virus in the supernatant. Therefore, we didn’t include any control in subsequent procedures (in ultra-centrifugation). However, we used media-only negative control in the titration results. We hope it will be enough to understand the results of this figure 2.

2. In Figure 4, I understand that the DENV-2 strain does not produce plaque. Is it possible to conduct non-CPE based neutralization assay such as FRNT?

Binding result by itself can be less scientifically sound.

             We only provided only qPCR data because our denv2 didn’t produce CPE. We tried PRNT several times for neutralization with the available serum, but it was not successful.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have answered most of my comments.  However, this manuscript still needs improvement before it can be considered for publication. My suggestions/comments are below-

Page 3, Table 1: The authors designed the sequence of the primers, or it was used from previous published studies. Please explain. If used from the previous studies, provide references.

Page 5, line 186. “Mice were immunized three times at 2 weeks intervals followed by blood was collected by retro-orbital bleeding 1 and 4 weeks after the 3rd booster” In these lines the authors mentioned they immunized mice three times, but Figure 4a shows immunization was done twice.  Please clarify.

Why the mice group that received the Envelope group showed IgG response similar to PBS? I agree authors have not used any adjuvant but still, the titer of IgG should be higher in mice that received adenovirus with envelope (it is a highly immunogenic protein) than mice that received PBS. What is the explanation of this?

Grammatical errors are detected throughout the manuscript. Please correct.

Page 1, lines 18 and 19: Purified adenoviruses (8 × 10^6 PFU/mL) were immunized administered intramuscularly to 6-week-old BALB/c mice. The DENV2-specific IgG titer was then evaluated at 1- and 4-weeks post-immunization.

Page 1, line 20- “Transduction efficiency of the generated adenoviruses was confirmed in A549 cells.” This line should be in line 17 (Before the mice experiment).

In the abstract authors have mentioned A549 cell lines but in the material and methods section (page 2, lines 76-80) A549 is not mentioned.

Page 3, line 109, “To generate of rAds for DENV2 virus envelope (E) and envelope ferritin heavy chain” remove OF.

Page 1, line 19-“Purified adenoviruses (8 × 106 PFU/mL) were immunized intramuscularly into 6 weeks old BALB/c mice”

Page 5, line 181-“Five-week-old Female BALB/c mice were maintained in the animal facility of Chung-National University (CNU), Korea”

Which one is correct? Six-week-old or five-week-old?

Comments on the Quality of English Language

Improvement is needed. Grammar error was found throughout the manuscript.

Author Response

Responses to Reviewer Report: Reviewer: 2

Page 3, Table 1: The authors designed the sequence of the primers, or it was used from previous published studies. Please explain. If used from the previous studies, provide references.

Response- we apologize for the missed information. Primer 1 and primer 2 sequences were obtained from a previous reference and the reference for the primers was included in the manuscript (lines 105-106 reference no. 25).

Page 5, line 186. “Mice were immunized three times at 2 weeks intervals followed by blood was collected by retro-orbital bleeding 1 and 4 weeks after the 3rd booster” In these lines the authors mentioned they immunized mice three times, but Figure 4a shows immunization was done twice.  Please clarify.

Response – We apologize for the misleading content. We immunized mice two times. The correct information was included in the manuscript (lines 190-191).

Why the mice group that received the Envelope group showed IgG response similar to PBS? I agree authors have not used any adjuvant but still, the titer of IgG should be higher in mice that received adenovirus with envelope (it is a highly immunogenic protein) than mice that received PBS. What is the explanation of this?

Response – We understand the reviewer’s curiosity about the lower immune response of envelope adenoviruses. Although it is essential to use high viral titer for immune response, we immunized only 8 × 10⁶ PFU/mL of adenoviruses per mouse because the titers of the adenoviruses we used were 2.60 × 10¹⁰ PFU/ mL and 1.77 × 10⁸ PFU/mL and 8 × 10⁶ PFU/mL was the maximum potential titer to be used for all 2 immunizations. We believe that if we increased the virus titer, the envelope-only group also could make IgG immune response and we have some evidence for that.

We immunized two mice with higher titer (1 × 10⁷ PFU/mL) of adenoviruses other than main experiment and it could increase the immune response of envelope only group compared to the lower titer (8 × 10⁶ PFU/mL) immunized. The relevant data is included below.

Grammatical errors are detected throughout the manuscript. Please correct.

Page 1, lines 18 and 19: Purified adenoviruses (8 × 10^6 PFU/mL) were immunized administered intramuscularly to 6-week-old BALB/c mice. The DENV2-specific IgG titer was then evaluated at 1- and 4-weeks post-immunization.

Response - We corrected the mentioned grammar mistake (line 21-22)

Page 1, line 20- “Transduction efficiency of the generated adenoviruses was confirmed in A549 cells.” This line should be in line 17 (Before the mice experiment).

Response - We changed the sentence location (before mice experiment) as requested (line 20).

In the abstract authors have mentioned A549 cell lines but in the material and methods section (page 2, lines 76-80) A549 is not mentioned.

Response -The cell and culture information were indicated in the materials and methods section (line 82-83).

Page 3, line 109, “To generate of rAds for DENV2 virus envelope (E) and envelope ferritin heavy chain” remove OF.

Response -We removed ‘OF’ from the mentioned sentence (line 113)

Page 1, line 19-“Purified adenoviruses (8 × 106 PFU/mL) were immunized intramuscularly into 6 weeks old BALB/c mice”

Page 5, line 181-“Five-week-old Female BALB/c mice were maintained in the animal facility of Chung-National University (CNU), Korea”

 Which one is correct? Six-week-old or five-week-old?

Response -We apologize for the misleading information. We used 6 weeks old Balb/C mice. The mic information was corrected as requested (line 21 and 185)

Author Response File: Author Response.docx