DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice

Eakachai Prompetchara; Chutitorn Ketloy; Kittipan Tharakhet; Papatsara Kaewpang; Supranee Buranapraditkun; Teerasit Techawiwattanaboon; Suwitra Sathean-anan-kun; Patrawadee Pitakpolrat; Supaporn Watcharaplueksadee; Supaporn Phumiamorn; Wassana Wijagkanalan; Kanitha Patarakul; Tanapat Palaga; Kiat Ruxrungtham

doi:10.1371/journal.pone.0248007

Peer Review History

Original SubmissionDecember 7, 2020
7 Jan 2021 Decision Letter - Paulo Lee Ho, Editor PONE-D-20-38402 DNA vaccine encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice PLOS ONE Dear Dr. Ketloy, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process: 1) Please, consider changing the title as suggested by reviewer #2; 2) The authors should provide more methodological details in order to allow others to repeat and confirm the results; 3) Please, answer to all the comments raised by the both reviewers. Please submit your revised manuscript by Feb 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1.) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.) Thank you for including your ethics statement: "Animal experiments were performed in strict accordance with the recommendations of the Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes. Mouse experimental procedures and animal management were undertaken in accordance with the requirement of the Committee of Animal Care and Use, Faculty of Medicine, Chulalongkorn University (approval number 007/2563). Immunization and blood collections were performed under isoflurane anesthesia. All efforts to minimize the suffering of the animals were made throughout the study. Please amend your current ethics statement to confirm that your named ethics committee specifically approved this study. For additional information about PLOS ONE submissions requirements for ethics oversight of animal work, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). 3.) In your Methods section, please provide methods of animal sacrifice and efforts to alleviate suffering. 4.) Thank you for stating the following in the Competing Interests section: 'This study was funded by National Vaccine Institute (NVI), grant no.2563.1/8, National Research Council of Thailand (NTCT), Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University, grant no RA-MF-29/63 and Second Century Fund (C2F), Chulalongkorn University. EP was also supported by the Grants for Development of New Faculty Staff, Ratchadapiseksompote Endowment Fund, grant no DNS 63_031_30_009_2.' We note that one or more of the authors are employed by a commercial company: BioNet-Asia Co., Ltd, a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 5.) PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The present study report results obtained by DNA vaccination using 3 different proteins from SARS-COV19 Spike – S (whole protein), S1 and S2. Antibodies and cellular response were generated, showing better results for the whole protein construction. The manuscript is well written, the results are expected but they are not novelty, comparing with other vaccine studies using SARS-COV 19 spike protein. DNA vaccines are easy to obtain and produce, are stable and stimulates both arms of immune response. It is well known. However, they still have technical problems that have not been solved until today – DNA vaccines working very well in murine models with results that are not repeated in primates, human and non-human. So far, we only have DNA vaccines approved for veterinary use. With these considerations, DNA vaccines at the moment serve more as proof of principle, which in this case (SARS-COV19) is already well known with other vaccine models. Other concerns Material and methods are poorly described. Reagent quantities are not described in several protocols; necessary corrections must be made. A methodology needs to be well described in order to allow other researchers to be able to reproduce them. Line 122 - Plasmids transfection and in vitro protein expression analysis - Protocols must be better described. Please, inform with more details the quantities of reagents were used in this assays. How much cells were transfected??? How much plasmid, lipofectamine and IgG-FITC were used??? Line 131 Please include the average weight of the animals. Were the blood samples taken on the same day as the immunizations? Please clarify. Line 140 - ELISA: Please, inform antigen concentrations in micrograms/well used. How much of sera was used, 100 microliters? Please, specify. Please inform quantity (titer) of HRP anti-mouse IgG was used. How much of TMB was used??? Line 158 - RBD-ACE2 binding inhibition How much of diluted mice sera (what dilution???) and RBD-HRP were used??? TMB??? What volume was used??? Line 282 : Please, describe what NHP means (non human primates???) Reviewer #2: Prompetchara et al describe the production and the use of plasmids containing full-length, just the S1, or S2 portions of the S1-glycoprotein of SARS-Cov2 in generating immune responses in mice. Similar studies have shown the possibility to generate immune responses in mice, guinea pig and Rhesus monkey. Recently, one of them has been adapted to be used in humans (https://doi.org/10.1016/j.eclinm.2020.100689), which could be additionally cited by the authors. The value of these manuscript could be to help in devising alternative ways to generate a protective response against the virus for human subjects and be used for vaccination. The results show improved responses with the use of full length spike, which should be better highlighted in the manuscript. Minor issues I would replace the title for DNA plasmid as there is not in vivo challenging in this work. Otherwise, use vaccine candidate. Line 38: define GMT in the abstract Line 40: stimulation Methods Plasmid nucleotide sequence details or deposit in public data bank should be provided. It is remarkable that many works have not disclosed this information, which I fell incorrect in scientific publications. The reference of pCMVkan should be cited in methods. Details about transfections (amounts and purification of DNA plasmids) are important to be mentioned here. In house preparation of S2 should be further explained. Results Fig. 2: size bars are poorly visible and Fig. 3: It is not clear which assay was used for IgG1xIgG2, please explain. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0248007.r001
Revision 1
27 Jan 2021 Author Response Re- PONE-D-20-38402 Dear Editor, Thank you very much for the useful comments from editor and reviewers. On behalf of the authors, I’d like to respond as following: Editor comments: 1. Please, consider changing the title as suggested by reviewer #2; Response: the title was revised to “DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice” 2) The authors should provide more methodological details in order to allow others to repeat and confirm the results; Response: See the responses on the methodological details to both Reviewer#1 and Reviewer#2 below 3) Please, answer to all the comments raised by the both reviewers. Response: See the response to both Reviewer#1 and Reviewer#2 below Reviewer#1 • Line 122 - Plasmids transfection and in vitro protein expression analysis - Protocols must be better described. Please, inform with more details the quantities of reagents were used in this assays. How much cells were transfected??? How much plasmid, lipofectamine and IgG-FITC were used??? Response: The details for plasmid transfection and in vitro protein expression was described. “At 24 hr before transfection, 1x106 cells of HEK293T were seeded in 6-well plate (Thermo Fisher Scientific, MA, USA). The cells with approximately 80-90% confluency were separately transfected with individual recombinant plasmid constructs (pCMVkan-S, -S1 and -S2) using lipofectamine 3000™ (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. Briefly, 2.5 µg of plasmid and 3.75 µL lipofectamine 3000™ were separately diluted in 125 µL and 250 µL serum-free Opti-MEM™ medium (Gibco), respectively. P3000™, a transfection enhancer reagent provided in lipofectamine 3000™ kit, was also added into diluted plasmid tube to a final concentration of 2 µL/µg DNA. The diluted DNA was mixed with the diluted lipofectamine 3000™ at 1:1 ratio (v/v) and incubated for 15 min at room temperature. The plasmid-lipofectamine 3000™ complex was then added onto cells.” (Line 136-146) • Line 131 Please include the average weight of the animals. Response: Mouse weight (20-25 grams) was included (Line 153). • Were the blood samples taken on the same day as the immunizations? Please clarify. Response: The detail for blood sample collection was clarified (Line 160-161). “Blood samples were collected at the day before each immunization, and 2 and 4 weeks after the 3rd dose designated as weeks 0, 2, 4, 6 and 8.” • Line 140 - ELISA: Please, inform antigen concentrations in micrograms/well used. How much of sera was used, 100 microliters? Please, specify. Please inform quantity (titer) of HRP anti-mouse IgG was used. How much of TMB was used??? Response: The detail for ELISA protocol was provided in the method including - antigen concentrations in micrograms/well used “In each well, the mixture of S1 (110 ng) and S2 (85 ng) which correspond to approximately 1.5 picomolar of each protein was diluted in 100 µL of coating buffer” (Line 177-178) - amount of sera “25 µL” (Line 182) - quantity (titer) of HRP anti-mouse IgG “1:5000 dilutions of each horseradish peroxidase (HRP)-conjugated secondary Abs” (Line 183) - Amount of TMB used 100 µL of tetramethylbenzidine (TMB) substrate (Line 186) • Line 158 - RBD-ACE2 binding inhibition How much of diluted mice sera (what dilution???) and RBD-HRP were used??? Response: The detail for RBD-ACE2 binding inhibition protocol was provided in the method including - dilution of sera diluted mice sera (1:100) (Line 195) - the use of RBD-HRP RBD conjugated with HRP (Line 195) - Volume of TMB used 100 µL of TMB (Line 197) - Volume of stop solution 50 µL of stop solution (Line 198) • Line 282 : Please, describe what NHP means (non human primates???) Response: Yes, NHP is stand for non-human primates (Line 316) Reviewer #2: • Prompetchara et al describe the production and the use of plasmids containing full-length, just the S1, or S2 portions of the S1-glycoprotein of SARS-Cov2 in generating immune responses in mice. Similar studies have shown the possibility to generate immune responses in mice, guinea pig and Rhesus monkey. Recently, one of them has been adapted to be used in humans (https://doi.org/10.1016/j.eclinm.2020.100689), which could be additionally cited by the authors. Response: The recent study in phase I clinical is cited “The study in phase I clinical trial demonstrated that DNA vaccine using full-length wild type spike was safe and could induce both antibodies and T cells responses (59)” (Ref 59, Line 388-390) • The results show improved responses with the use of full length spike, which should be better highlighted in the manuscript. Response: We highlighted the use of wild-type full length which it could provide high level of protective efficacy as seen in ChAdOx1 nCoV-19 (in discussion Line 392-396). And based on our findings, in conclusion we also state that full length S (S1+S2) is better than the truncated S1 or S2 immunogens (Line 408-401). • I would replace the title for DNA plasmid as there is not in vivo challenging in this work. Otherwise, use vaccine candidate. Response: the title was revised to “DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice” • Line 38: define GMT in the abstract Response: Geometric mean titers added (Line 49) • Line 40: stimulation Response: corrected to stimulation (Line 52) • Plasmid nucleotide sequence details or deposit in public data bank should be provided. It is remarkable that many works have not disclosed this information, which I fell incorrect in scientific publications. Response: As mentioned in method section, we used the sequence from online database which is the wild-type sequence without further modification of amino acids. So, the sequence still the same with the uploaded database. • The reference of pCMVkan should be cited in methods. Response: The information of pCMVkan and reference was included “pCMVkan expression vector which contain the cytomegalovirus promoter, the bovine growth hormone polyadenylation site, and the kanamycin-resistant gene (24)” (Ref 24, Line 125-127) • Details about transfections (amounts and purification of DNA plasmids) are important to be mentioned here. Response: The details for plasmid transfection and in vitro protein expression was described “At 24 hr before transfection, 1x106 cells of HEK293T were seeded in 6-well plate (Thermo Fisher Scientific, MA, USA). The cells with approximately 80-90% confluency were separately transfected with individual recombinant plasmid constructs (pCMVkan-S, -S1 and -S2) using lipofectamine 3000™ (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s protocol. Briefly, 2.5 µg of plasmid and 3.75 µL lipofectamine 3000™ were separately diluted in 125 µL and 250 µL serum-free Opti-MEM™ medium (Gibco), respectively. P3000™, a transfection enhancer reagent provided in lipofectamine 3000™ kit, was also added into diluted plasmid tube to a final concentration of 2 µL/µg DNA. The diluted DNA was mixed with the diluted lipofectamine 3000™ at 1:1 ratio (v/v) and incubated for 15 min at room temperature. The plasmid-lipofectamine 3000™ complex was then added onto cells.” (Line 136-146), Purification of DNA plasmids procedure was mentioned “The recombinant plasmids were propagated in E. coli, DH5-alpha (Invitrogen, Carlsbad, CA, USA) and purified by Qiagen endotoxin-free giga plasmid kit (Hilden, Germany) following the manufacturer’s protocol.”(Line 130-133) • In house preparation of S2 should be further explained. Response: The procedure of S2 production and purification is described “The S2 protein was prepared by induction of the plasmid pET15b encoding S2 region fused with 6HIS at N terminal. In brief, transformed E. coli BL21(DE3) were grown at 37°C in LB (Luria-Bertani) medium supplemented with 100 µg/mL ampicillin. When the OD600 of the cell culture reached 0.6, isopropyl β-D-1-thiogalactopyranoside, IPTG (final concentration 0.1 mM) was added to induce protein expression and cells were grown at 25°C for a further 3 h. Cells were harvest by centrifugation at 8,000 g for 10 min at 4°C. For S2 purification, the cell pellet was resuspended in PBS supplemented with DNase (10 µg/mL) and RNase (10 µg/mL). The cells were lysed by sonication on ice for 15 min. The lysed cells were then centrifuged at 40,000 rpm for 1 h, only supernatant was collected. S2 protein was purified by affinity chromatography using HisTrap column (GE Healthcare, Sweden). The concentration of purified protein was measured by using Pierce™ BCA protein assay kit (Thermo Fisher Scientific, MA, USA).”(Line 165-176) • Fig. 2: size bars are poorly visible and Response: The bar size was improved • Fig. 3: It is not clear which assay was used for IgG1xIgG2, please explain. Response: IgG1 and IgG2a were analyzed by ELISA using secondary antibodies which specific to IgG1 and IgG2a subclasses. The method is mention in Line 182-185. “The plates were then washed and incubated with 1:5000 dilutions of each horseradish peroxidase (HRP)-conjugated secondary Abs including rabbit anti-mouse IgG (KPL, USA), rabbit anti-mouse IgG1 or rabbit anti-mouse IgG2a (both Abs were from BioLegend, USA) for additional 1 h at 37°C.” Attachments Attachment Submitted filename: Response to reviewers PONE-D-20-38402.docx https://doi.org/10.1371/journal.pone.0248007.r002
11 Feb 2021 Decision Letter - Paulo Lee Ho, Editor PONE-D-20-38402R1 DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice PLOS ONE Dear Dr. Ketloy, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process: 1) Please, provide the gene sequence as supplemental information as requested by reviewer #2 Please submit your revised manuscript by Mar 28 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: After read, the last version, I concluded that all the comments were addressed, the manuscript is ready for publication. Reviewer #2: Most of the required details were provided. However, the authors should provide a DNA sequence of the construct used for expression. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes:** Sergio Schenkman [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0248007.r003
Revision 2
11 Feb 2021 Author Response As the reviewer suggested the authors to provide a DNA sequence of the construct used for expression, the nucleotide sequences (humanized codons) of Spike (S), S1 and S2 are attached as supplementary data 1 (line 128). Attachments Attachment Submitted filename: Response to reviewers PONE-D-20-38402 R2.docx https://doi.org/10.1371/journal.pone.0248007.r004
18 Feb 2021 Decision Letter - Paulo Lee Ho, Editor DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice PONE-D-20-38402R2 Dear Dr. Ketloy, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0248007.r005
Formally Accepted
10 Mar 2021 Acceptance Letter - Paulo Lee Ho, Editor PONE-D-20-38402R2 DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice Dear Dr. Ketloy: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Paulo Lee Ho Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248007.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .