Peer Review History

Original SubmissionJuly 18, 2019
Decision Letter - Ana Fernandez-Sesma, Editor, Mehul Suthar, Editor

Dear Dr. Akbari:

Thank you very much for submitting your manuscript "Broad Dengue Neutralization in Mosquitoes Expressing an Engineered Antibody" (PPATHOGENS-D-19-01283) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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[LINK]

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Mehul Suthar

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors present new genetically engineered Ae. aegypti strain expressing a highly and broadly cross-neutralizing single chain antibody against DENV. They demonstrate through a series of experiments that these engineered mosquitoes can effectively (100%) inhibit the infection, dissemination and transmission of all four serotypes of DENV. Further, they demonstrate that their strain is even more effective than Wolbachia, which is currently the gold standard for DENV inhibition. Overall this is a very exciting and impactful manuscript. Below are my critiques.

Reviewer #2: The study by Buchman et al generated Aedes aegypti mosquitoes that produce a single chain antibody that is capable of neutralizing all four strains of Dengue virus. The paper is well written and contains necessary controls and experiments for the conclusions they are drawing. They engineered the antibody expression to coincide with blood meal digestion to limit expression and find it had little impact on mosquito fitness. Mosquitoes homozygous for the antibody appear completely refractory for virus whereas heterozygotes tested positive for virus, but contained significantly lower quantities of virus. They include Wolbachia infected mosquitoes to compare their results with other well-established strategies to decrease mosquito competence for Dengue transmission. They find that the heterozygotes carrying the antibody decrease viral quantities similar to Wolbachia infected mosquitoes and homozygotes are more effective. While outside of this particular study, I cannot help but wonder how quickly Dengue will be able to escape neutralization from a single antibody. This is a very provocative study demonstrating a new method to decrease dengue spread. Future work would require incorporating antibody expression with a potent gene drive.

Reviewer #3: This is a very elegant study where transgenic Aedes aegypti expressing an engineered single-chain variable fragment derived from a broadly neutralizing dengue virus human monoclonal antibody are refractory to all four dengue virus serotypes. As such the study can be considered a significant advance that paves the way to develop transgenic mosquito-based dengue control. One weakness is the lack of basic biological insight.

Reviewer #4: Strengths: This paper by Buchman et al builds on previous work by the group where they engineered miRNAs in Aedes aegypti for resistance to DENV1-4. In the paper under review, they have engineered Mab 1C19 as a single-chain variable fragment (scFv) to build an effector transgene for resistance to multiple DENV serotypes. The key innovative aspect of this work is the development of the scFv from IC19 since the monoclone has broad neutralzing activity against the four serotypes. This type of approach (targeting all four serotypes) has been a 'holy grail' of geneticists/arbovirologists interested in designing Aedes aegypti for resistance to DENVs and furthers population replacement strategies/approaches. This research group also has a continued interest in developing antiviral transgenes in the context of gene drive so this might all come together. An important feature of this work is the IC19 scFv expression levels are increased with homozygosity generating apparent complete resistance to DENV1-4. It was a nice touch to compare DENV resistance of TADV-A vs wMel Wolbachia infected mosquitoes. The paper is well written and follows a logical experimental approach for developing transgenesis and characterizing transgene efficiency and a DENV 1-4 resistance phenotype.

Weaknesses: The weaknesses are minor. However, the approach brings up two points. First, in theory minor changes in the viral genome at the target site could reduce or ablate resistance by altering the ability of the scFv to neutralize. The authors discussed this in their paper and thought they could manage this outcome through selection of other scFVs transgenes, but these may run into the same problem. Nevertheless this approach may drive evolution/selection of viruses that fail to interact with these transgene effectors. Perhaps building transgenics with multiple transgenes may mitigate the ability of DENVs to adapt to any one transgene and designing a mosquito with a combination of effector gene approaches may be a future goal. Second, the fitness of the TADV-A may be further improved by introgressing the scFv transgene into a field-collected (true wild-type) strain of Aedes aegypti since the progenitor HWE strain contains recessive lethals that compromise fitness. However, I view these as minor issues for this paper. Please address.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #3: The authors could have elaborated more on biological aspects, such as the RNAseq data (unlikely that no endogenous mosquito gene were not regulated). The authors should also elaborate on the fitness data that did show some interesting patterns especially for dengue virus 1.The comparison between the transgenic mosquitoes and Wolbachia only involved one virus strain (a lab strain) so one should be cautious with the interpretation of that data.

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. The following excerpt is a run-on sentence and should be changed. Line numbering would have been helpful.

“Moreover, releases of mosquitoes artificially infected with the intracellular endosymbiont Wolbachia, which can make infected males incapable of successfully mating with uninfected females in an SIT-like manner and can inhibit mosquito infection with pathogens such as DENV and ZIKV(Aliota et al., 2016;Walker et al., 2011), have also been carried out to either suppress mosquito populations or make them less likely to transmit pathogens, and may hold promise for reducing incidence of disease(Moreira et al., 2009; Schmidt et al., 2017).”

2. A description of the carboxypeptidase promoter should be presented. Presumably, it is constitutively expressed and only translated following a bloodmeal? This should be included somewhere in the manuscript.

3. It is unclear where this scFv is expressed. Is it secreted, membrane bound or cytosolic? There was no mention of its localization. If it is secreted how does the antibody bind the virus prior to infection? Presumably, transcripts will be translated upon bloodfeeding (see above comment), but temporally how does this align with the rate of midgut infection? Can the antibodies still block infection after internalization into early endosomes? I am not suggesting this paper needs to be converted into a mechanistic paper, but it should discuss these questions.

4. The first paragraph of the discussion discusses how heterozygous mosquitoes could be sufficient to halt transmission due to their reduction in transmission. This may or may not be true which is acknowledged; however, if this argument that heterozygous mosquitoes could be effectively used in nature if needed than it would have been nice to see the fitness studies expanded to include the TADV-A heterozygotes.

5. The authors discuss the possibility of escape mutants in the discussion. I don’t think this is a major concern in the homozygotes because they demonstrated sterilizing immunity; however, this could be important in heterozygotes.

Reviewer #2: Minor:

What was the concentration of virus in the blood-meal? I was unable to find this information in the manuscript.

Sup Fig 1: Western blots for the Non blood-fed midguts should be run on the same gel as the positives to demonstrate relative expression levels. Running the samples on multiple gels makes things harder to evaluate and potentially makes the background bands more apparent.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Revision 1

Attachments
Attachment
Submitted filename: PLOS Pathogens Rebuttal.docx
Decision Letter - Ana Fernandez-Sesma, Editor, Mehul Suthar, Editor

Dear Dr. Akbari,

We are pleased to inform that your manuscript, "Broad Dengue Neutralization in Mosquitoes Expressing an Engineered Antibody", has been editorially accepted for publication at PLOS Pathogens. 

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. 

Best regards,

Mehul Suthar

Associate Editor

PLOS Pathogens

Ana Fernandez-Sesma

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

***********************************************************

Reviewer Comments (if any, and for reference):

Formally Accepted
Acceptance Letter - Ana Fernandez-Sesma, Editor, Mehul Suthar, Editor

Dear Dr. Akbari,

We are delighted to inform you that your manuscript, "Broad Dengue Neutralization in Mosquitoes Expressing an Engineered Antibody," has been formally accepted for publication in PLOS Pathogens.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

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