Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus

He Sun; Guangmou Wu; Jiyuan Zhang; Yu Wang; Yue Qiu; Hongyang Man; Guoli Zhang; Zehong Li; Yuhuan Yue; Yuan Tian

doi:10.1371/journal.pone.0266220

Peer Review History

Original SubmissionNovember 5, 2021
16 Dec 2021 Decision Letter - Hari S. Misra, Editor PONE-D-21-35307Characterization of An Intracellular Humanized Single-Chain Antibody to Matrix Protein (M1) of H5N1 VirusPLOS ONE Dear Dr. Yuhuan, 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. Please submit your revised manuscript by Jan 30 2022 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. 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Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Dear Dr Yue Yuhuan, Thank you for submitting your work to PLoS ONE. This has been reviewed by 2 experts in the field. Both of them have appreciated it but have also suggested some work for further improvement. I suggest to revise manuscript it by addressing all the concerns of both the reviewers. [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A ****** 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: No Reviewer #2: Yes ****** 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: This study by He Sun et. al. reports a novel single chain humanized antibody fragment against the M1 protein of the avian influenza virus generated using phage library. The methodology is well described, and the results are well summarized. The manuscript needs further improvement including sharing of raw data for the results presented and an overall copy editing. Refer to the queries listed below. Major comments: • This study reports multiple ELISA assays, however the results of these assays (images of the plates at the least or the actual read outs) have not been provided, the absence of which makes the study look superficial and non-authentic. • Similarly for the statement “The purified TAT-HuScFv and HuScFv were compared for hemagglutination inhibition; the ability of TATHuScFv when fused to the TAT domain to bind viral M1 protein was stronger than that of HuScFv (Table 3)”, no actual data has been presented in the manuscript, making the interpretations vague. • The authors need to examine the efficacy of the TAT-HuScFv and HuScFv fragments in neutralizing/inhibiting viral infection in a suitable ex vivo infection model. Minor Comments: • Rephrase the sentence “The 16 sub-types of hemagglutinin HA (H1-H16) and the nine sub-types of neuraminidase NA (N1-N9) are proteins involved in avian influenza” to express it more clearly. • Provide a reference for the statement “The pET28a-TAT-GFP vector was constructed previously by our research group.” • What was the nature of competency in the BL 21 cells mentioned in the sentence “The vector was sequenced and then transformed into BL21 (DE3) competent cells.” • Replace “eluded” with “eluted” in the phrase “The eluded phage was cloned into E. coli TG1”. • Rephrase the sentence “The pET28A-TAT-HuScFv construct was then transformed into BL21(DE3) with the CaCl2 method.” to “The pET28A-TAT-HuScFv construct was then transformed into chemically competent BL21(DE3) cells.” • Rephrase this sentence “The supernatant of the expressed cells was obtained following ultrasonic lysis”. I believe the authors intend to say cells expressing the TAT-HuScFv and the supernatant was obtained post lysis and centrifugation of the lysate. • Rephrase “The supernatant of cells was then absorbed”. • Rephrase and expand to make this statement clear “Analysis involved 3, 3′,5 ,5′- Tetramethylbenzidine (TMB) color and the positive fragments.” • Rephrase “this was the expected size of the fusion protein” as “which was the expected size of the fusion protein”. • Rephrase with a clearer description “Two polypeptides, containing eight amino acids (QA) were synthesized; then, Q A was genetic mutated into E (ENLEAYQK) G(ENLEQGYQK) respectively.” • Penetrate instead of “penetrat” in the sentence “we demonstrated that TAT-HuScFv can quickly penetrat the cell”. • The main text includes a lot of complex sentences (often joined using semi-colon), making it hard to read. I suggest the author perform a careful copy-editing of the manuscript and reduce such sentences. Reviewer #2: Author Sun et. al., provides well characterized humanized antibodies against M1 protein, which showed excellent anti-influenza activity as shown by hemagglutination assay and found out that the single amino acid change may alter the binding. Here are my comments: Abstract section: 1- Change the sentence to “cDNA of the H5N1 virus as a template; the M1 protein was then expressed and purified” to ‘cDNA of the H5N1 virus as a template, expressed in bacterial expression system (name the bacteria used) and purified.’ 2- Space in 300 TCID50 line no 09; against the; line no 12. 3- Please use correct grammar and sentences in writing. Method section: 1- First para line-3, provide the reference. 2- If virus was used in the study, please provide, the preparation method, stock concentration estimation etc. 3- Whether PCR amplicon of M1 was PCR/gel purified and any restriction enzymes were used to ligate into bacterial vector. 4- Please provide purification method for M1 protein 5- Please re-write this long sentence ‘Next, the Tomlison I+J phage antibody library was added and diluted with 2% milk/PBS to a titer of 1.0 ×1013; 100 μL was added to each well, and the liquid was incubated with vigorous shaking at room temperature for 60 min.’ 6- WB analysis paragraph, used probed instead of ‘blotted’ 7- Add extension time of PCR in Expression and Purification of TAT-HuScFv paragraph 8- First line of Hemagglutination Inhibition Analysis of Anti-M1-HuScFv and TAT-HuScFv ‘Digested MDCK cells’ with which enzyme? 9- Line 1-2; page no 12; please highlight or bold the changed amino acid in the small peptides. 10- Provide the role of GFP in this construct pET 28 TAT- HuScFv. Results section: Please provide a full blot of figure-2B. In discussion, please provide the comparison of anti M1-TAT-HuScFv and previously published humanized antibodies against M1 protein. ****** 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: Yes:** RAVI P. ARYA [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. Attachments Attachment Submitted filename: Plosone_comments.docx https://doi.org/10.1371/journal.pone.0266220.r001
Revision 1
3 Mar 2022 Author Response Dear editor: Thank you very much for your comments and I will reply one by one here. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. I understand. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. We received two grants, Jilin Province Science and technology development plan. 20200404113YY, 20190304038YY, Please find the attached file A. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. No changes need to be made. 4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. I understand. 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 I understand. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. I understand. After my careful review, there are no retracted articles. Reviewer #1: 1. This study reports multiple ELISA assays, however the results of these assays (images of the plates at the least or the actual read outs) have not been provided, the absence of which makes the study look superficial and non-authentic. We can provide real data for your review. Please find the attached file B. 2. Similarly for the statement “The purified TAT-HuScFv and HuScFv were compared for hemagglutination inhibition; the ability of TATHuScFv when fused to the TAT domain to bind viral M1 protein was stronger than that of HuScFv (Table 3)”, no actual data has been presented in the manuscript, making the interpretations vague. It can be clearly seen from Table 3 that in the comparison of hemagglutination inhibition between TAT-HuScFv and HuScFv, the results of hemagglutination of TAT-3F and TAT-7B are better than those of 3F and 7B. 3. The authors need to examine the efficacy of the TAT-HuScFv and HuScFv fragments in neutralizing/inhibiting viral infection in a suitable ex vivo infection model. What you said is very correct and agrees with your point of view. This is exactly what our research group is doing now, and we will work hard to improve it. My classmate is working on the titer of TAT-HuScFv in the mouse model. 4. Rephrase the sentence “The 16 sub-types of hemagglutinin HA (H1-H16) and the nine sub-types of neuraminidase NA (N1-N9) are proteins involved in avian influenza” to express it more clearly. I understand. 5. Provide a reference for the statement “The pET28a-TAT-GFP vector was constructed previously by our research group.” This vector was constructed by the senior sister Xu Yanling from the previous research group and published in the Chinese core journal "Chinese Journal of Biological Products"-Purification of PTD-GFP fusion protein and determination of its transduction efficiency. Please find the attached file C. 6. What was the nature of competency in the BL 21 cells mentioned in the sentence “The vector was sequenced and then transformed into BL21 (DE3) competent cells.” BL21 (DE3) competent cells were obtained by using escherichia coli BL21 (DE3) strain through special processing, which can be used for chemical transformation of DNA. BL21 (DE3) strain was suitable for expressing non-toxic proteins, and the strain was the host of high expression of exogenous gene proteins with T7 RNA polymerase as expression system. Using pU19 plasmid detection, the conversion efficiency can reach 107, and the conversion efficiency does not change when stored at -80℃ for a long time. 7. Replace “eluded” with “eluted” in the phrase “The eluded phage was cloned into E. coli TG1”. I understand. 8. Rephrase the sentence “The pET28A-TAT-HuScFv construct was then transformed into BL21(DE3) with the CaCl2 method.” to “The pET28A-TAT-HuScFv construct was then transformed into chemically competent BL21(DE3) cells.” I understand. 9. Rephrase this sentence “The supernatant of the expressed cells was obtained following ultrasonic lysis”. I believe the authors intend to say cells expressing the TAT-HuScFv and the supernatant was obtained post lysis and centrifugation of the lysate. I understand. 10. Rephrase “The supernatant of cells was then absorbed”. I understand. 11. Rephrase and expand to make this statement clear “Analysis involved 3, 3′,5 ,5′- Tetramethylbenzidine (TMB) color and the positive fragments.” I understand. 12. Rephrase “this was the expected size of the fusion protein” as “which was the expected size of the fusion protein”. I understand. 13. Rephrase with a clearer description “Two polypeptides, containing eight amino acids (QA) were synthesized; then, Q A was genetic mutated into E (ENLEAYQK) G(ENLEQGYQK) respectively.” I understand. 14. Penetrate instead of “penetrat” in the sentence “we demonstrated that TAT-HuScFv can quickly penetrat the cell”. I understand. 15. The main text includes a lot of complex sentences (often joined using semi-colon), making it hard to read. I suggest the author perform a careful copy-editing of the manuscript and reduce such sentences. I understand. Reviewer #2: 1. Change the sentence to “cDNA of the H5N1 virus as a template; the M1 protein was then expressed and purified” to ‘cDNA of the H5N1 virus as a template, expressed in bacterial expression system (name the bacteria used) and purified.’ I understand. 2. Space in 300 TCID50 line no 09; against the; line no 12. I understand. 3. Please use correct grammar and sentences in writing. This manuscript has been edited by a native English professor with a doctorate degree and the International Science Editing corporation again. 4. First para line-3, provide the reference. I understand. 5. If virus was used in the study, please provide, the preparation method, stock concentration estimation etc. The H1N1 virus was passed by MDCK cells. After the MDCK cells were covered with a single layer, 2 mL DMEM medium containing H1N1 was added into 25 cm2 culture flask for incubation for 1 h, and 3 mL DMEM (containing 0.5% TPCK trypsin) was added, and the virus was recovered after 48 h. The virus TCID50 was 105.37 by Reed-Muench method. 6. Whether PCR amplicon of M1 was PCR/gel purified and any restriction enzymes were used to ligate into bacterial vector. The PCR amplicon of M1 was PCR/gel purified. And any restriction enzymes weren't used to ligate into bacterial vector. 7. Please provide purification method for M1 protein. I understand. 8. Please re-write this long sentence ‘Next, the Tomlison I+J phage antibody library was added and diluted with 2% milk/PBS to a titer of 1.0 ×1013; 100 μL was added to each well, and the liquid was incubated with vigorous shaking at room temperature for 60 min.’ I understand. 9. WB analysis paragraph, used probed instead of ‘blotted’. I understand. 10. Add extension time of PCR in Expression and Purification of TAT-HuScFv paragraph. I understand. 11. First line of Hemagglutination Inhibition Analysis of Anti-M1-HuScFv and TAT-HuScFv ‘Digested MDCK cells’ with which enzyme? Trypsin commonly used to digest cells. 12. Line 1-2; page no 12; please highlight or bold the changed amino acid in the small peptides. I understand. 13. Provide the role of GFP in this construct pET 28 TAT- HuScFv. The GFP tag has no effect, because the corresponding vector used is left over from a previous laboratory, and although its name can be seen in this study, it will not play any role. 14. Please provide a full blot of figure-2B. I understand. Please find the attached file D. 15. In discussion, please provide the comparison of anti M1-TAT-HuScFv and previously published humanized antibodies against M1 protein. I understand. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0266220.r002
17 Mar 2022 Decision Letter - Hari S. Misra, Editor Characterization of an Intracellular Humanized Single-Chain Antibody to Matrix Protein (M1) of H5N1 Virus PONE-D-21-35307R1 Dear Dr. Yuhuan, 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, Hari S. Misra Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0266220.r003
Formally Accepted
22 Mar 2022 Acceptance Letter - Hari S. Misra, Editor PONE-D-21-35307R1 Characterization of an Intracellular Humanized Single-Chain Antibody to Matrix Protein (M1) of H5N1 Virus Dear Dr. Yue: 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 Professor Hari S. Misra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0266220.r004

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