Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16

Chongzhi Bai; Ruoyu Wang; Qian Yang; Jianqing Hao; Qiming Zhong; Ruiwen Fan; Pengcheng Han

doi:10.1371/journal.pone.0311137

Peer Review History

Original SubmissionJuly 1, 2024
5 Aug 2024 Decision Letter - Haitham Mohamed Amer, Editor PONE-D-24-26930Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16PLOS ONE Dear Dr. Bai, 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 Sep 19 2024 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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This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. [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: I Don't Know ****** 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: Overview: This study aimed to engineer, express, purify, and characterize two immunogenic fusion proteins targeting HPV 16-L1 protein. The study further aims to compare it to 2C12, a nanobody previously generated by these investigators against the same target. Computational analysis was performed to characterize the residues critical to target binding further. Given that only prophylactic vaccines, and not therapeutic vaccines, are available for HPV, the work addresses an important need and the use of nanobodies seems to be a novel and scalable approach. Introduction The authors do a nice job of providing an introduction to nanobodies and summarizing their key findings. Weaknesses - For clarity and relevance to the current study, it would have been helpful to make a stronger case as to why a therapeutic vaccine would be important in the field and why current prophylactic vaccines are insufficient to address the public health issue of HPV-mediated cancers. An excellent review on this topic can be found at: https://doi.org/10.3389/fimmu.2024.1362770 Methods Can the authors provide additional detail? At present, there is insufficient detail to replicate the work in this report. The manufacturers of the reagents and analyzers used to collect data are not provided. For example, the authors used pColdII as the parent vector to create the fusion protein construct. Where did they obtain pColdII? For the IMAC purification, which resin did they use, how did they elute the Ni-column – no details or data are provided. The specific MW standards used were not described for the gel filtration chromatography and SDS PAGE. For the Biosensor experiments, the authors do not provide details of their assay, such as the concentration range and specific concentration of the fusion proteins for each analysis. They also don’t describe performing blank runs. Was this done? For the pseudovirus inhibition experiments, can the authors please provide more details on what the CQI microscopy reading method entails, including a reference? Also, how were the images obtained? Line 127 has a typo. Results Purification – the authors seem to have purified the fusion proteins to near homogeneity; however, the lack of specific details makes this hard to determine conclusively. The yield of the fusion proteins was not described. Figure 1. The gel filtration standards should be named, and their elution volumes should be indicated on the chromatogram. SDS PAGE across the gel filtration fractions should be provided to demonstrate that they correspond to AHPVP and AHPVT. What are the commercial names and providers of the MW standards used in the gel filtration and SDS-PAGE? Figure 1C. What percentage of gel is shown? How much protein is run in each lane. How are the bands detected? Figure 2. The blank run is not provided. I could not find the analyte concentrations used in each run on the sensograms. This information should be provided. What exactly is “Z6”? Please provide the numerical data that corresponds to the sensograms. Figure 3. The resolution and low signal make the images hard to interpret. Based on the curve shown in Figure 3E, there should be a significant difference between the images shown in Figure 3A and 3B – as shown, they seem identical. Also, the curves shown for AHPVD and AHPVT don’t seem to reflect the differences in the signal shown in the images. Figure 4. What templates were used to create the models in Alphafold2? Two different abbreviation conventions are used to name the amino acids in this one figure: the three-letter code and the one-letter code. The authors should stick to one approach. The average pLDDT values were not provided. Discussion The authors didn’t really discuss their work in the context of previous work done – they simply reiterated their results. Reviewer #2: 1. Justify the role of major capsid protein L1 with elaborative explanation and proper references. 2. The language and sentence making of methods section need to be polished. Few sentences are not clearly expressing the performed steps. 3. Why alphafold2 server was used, there are many other validated servers for structure prediction including Itasser, Trosetta etc. Justify the use of this specific server in this study? 4. The 3D structure of 2C12 was predicted using alphafold2, but further validation and quality assessment of the 3D structure is missing. A proper validation which includes ERRAT quality score, favored region in Ramachandran plot and z score need to be provided with figure. 5. Some of the explanation in discussion needs proper explanation. For example, this line “Molecular docking analysis using ZDock3.0.2 revealed that 2C12 and HPV16 L1 formed five pairs of hydrogen bonds as well as multiple hydrophobic interactions”, what is the significance of hydrogen bond and hydrophobic interaction. Appropriate justification with reference is vital. 6. Compare some of the findings with previous study which is missing in the discussion. Is there any similar study or findings exist for AHPVD and AHPVT, compare with other relevant studies? 7. Illustrate figure 4 more clearly, you can improve the quality and resolution of the picture. ****** 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". 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Revision 1
28 Aug 2024 Author Response Response to Reviewers 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: I Don't Know ________________________________________ 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: Overview: This study aimed to engineer, express, purify, and characterize two immunogenic fusion proteins targeting HPV 16-L1 protein. The study further aims to compare it to 2C12, a nanobody previously generated by these investigators against the same target. Computational analysis was performed to characterize the residues critical to target binding further. Given that only prophylactic vaccines, and not therapeutic vaccines, are available for HPV, the work addresses an important need and the use of nanobodies seems to be a novel and scalable approach. Introduction The authors do a nice job of providing an introduction to nanobodies and summarizing their key findings. Weaknesses - For clarity and relevance to the current study, it would have been helpful to make a stronger case as to why a therapeutic vaccine would be important in the field and why current prophylactic vaccines are insufficient to address the public health issue of HPV-mediated cancers. An excellent review on this topic can be found at: https://doi.org/10.3389/fimmu.2024.1362770 Response: Thank you for the comments and the literature sharing. We really appreciate your acknowledgement of our studies and also see your concerns in many aspects. As suggested, we have carefully read the literature and added the description that why a therapeutic vaccine would be important in the field and why current prophylactic vaccines are insufficient to address the public health issue of HPV-mediated cancers. (line 61-65) Methods Can the authors provide additional detail? At present, there is insufficient detail to replicate the work in this report. The manufacturers of the reagents and analyzers used to collect data are not provided. For example, the authors used pColdII as the parent vector to create the fusion protein construct. Where did they obtain pColdII? For the IMAC purification, which resin did they use, how did they elute the Ni-column – no details or data are provided. The specific MW standards used were not described for the gel filtration chromatography and SDS PAGE. Response: Thanks for the suggestion. We have provided the manufacturers of the reagents and analyzers used to collect data in the revised manuscript. (line 92-100 , line112-118 and line 205) For the Biosensor experiments, the authors do not provide details of their assay, such as the concentration range and specific concentration of the fusion proteins for each analysis. They also don’t describe performing blank runs. Was this done? Response: Thank you for your kind suggestion. We have provided details of the assay in the revised manuscript. (line 124-129) For the pseudovirus inhibition experiments, can the authors please provide more details on what the CQI microscopy reading method entails, including a reference? Also, how were the images obtained? Line 127 has a typo. Response: Thank you for your kind suggestion. We have provided details of the assay and revised the typo in the revised manuscript. (line 160-162) Results Purification – the authors seem to have purified the fusion proteins to near homogeneity; however, the lack of specific details makes this hard to determine conclusively. The yield of the fusion proteins was not described. Response: Thank you for your kind suggestion. Indeed, it seems hasty to draw this conclusion. We have revised the sentence as follows “Based on the comparison with the HiLoad 16/600 Superdex 75 prep grade protein standard curve, AHPVD and AHPVT eluted at about 70 min and 60 min, respectively, and he curve is symmetrical (Fig 1A and 1B). The single target protein bands corresponding to their theoretical molecular weights of 30 kD and 45 kD were observed on a 12.5% SDS-PAGE gel (Fig 1C). Combined with gel filtration chromatography and SDS-PAGE gel, we can infer that relatively homogeneous and highly purified proteins are obtained.” (line 200-208) Figure 1. The gel filtration standards should be named, and their elution volumes should be indicated on the chromatogram. SDS PAGE across the gel filtration fractions should be provided to demonstrate that they correspond to AHPVP and AHPVT. What are the commercial names and providers of the MW standards used in the gel filtration and SDS-PAGE? Response: Thank you for your kind suggestion. As you suggested, we have provided the details about the gel filtration chromatography in the “Materials and methods” part (line112-118), revised the figure legend and resubmitted Figure 1. The commercial names and providers of the MW standards used in the gel filtration and SDS-PAGE have been provided. (line93) Figure 1C. What percentage of gel is shown? How much protein is run in each lane. How are the bands detected? Response: Sorry, we didn't describe it clearly in the previous manuscript. We have revised this section. (line203-207) Figure 2. The blank run is not provided. I could not find the analyte concentrations used in each run on the sensograms. This information should be provided. What exactly is “Z6”? Please provide the numerical data that corresponds to the sensograms. Response: Thank you for your kind suggestion. As you suggested, We have provided details of the assay in the revised manuscript. (line 126-129) Z6 was used as a negative control, it is Anti-ZIKV human mAb, which was prepared and stored in our laboratory. (line 97-99) Figure 3. The resolution and low signal make the images hard to interpret. Based on the curve shown in Figure 3E, there should be a significant difference between the images shown in Figure 3A and 3B – as shown, they seem identical. Also, the curves shown for AHPVD and AHPVT don’t seem to reflect the differences in the signal shown in the images. Response: I'm sorry to have caused you trouble. In fact, the resolution of figure 3 meets the requirements. It may be due to the distortion of the resolution after uploads, which makes it impossible to identify. Please download the TIF format image to view. As described in the manuscript line 219-221 “AHPVT exhibited even higher neutralization activity with an IC50 value of 6.5 nM suggesting its superior binding ability in efficiently targeting multiple HPV16 virus particles”. In order to more clearly demonstrate the higher neutralizing activity of AHPVT and AHPVD than 2C12, we selected the pictures of the four proteins at 6.5 nM concentration Figure 3A-3D. The neutralization activity of four proteins at this concentration were approximately Z6 was no more than 5%, 2C12 was no more than 10%, AHPVD was 30%, AHPVT was 50%, respectively. From the images, it is difficult to obtain a clear difference between Figure 3A and 3B, also between Figure 3C and 3D. We have uploaded the data in the Supporting information. Figure 4. What templates were used to create the models in Alphafold2? Two different abbreviation conventions are used to name the amino acids in this one figure: the three-letter code and the one-letter code. The authors should stick to one approach. The average pLDDT values were not provided. Response: Thank you for your kind suggestion and sorry for the trouble brought to you in the review. We predicted the structure using default Settings and did not use templates. We have revised the Figure 4 as you suggested, and the average pLDDT values were 91.805(line 178-180). Discussion The authors didn’t really discuss their work in the context of previous work done – they simply reiterated their results. Response: Thank you for your kind suggestion. We have revised the manuscript. Reviewer #2: 1. Justify the role of major capsid protein L1 with elaborative explanation and proper references. Response: Thank you for your kind suggestion. We have revised the manuscript and added some descriptions of the role of major capsid protein L1. (line 51-53 and line62-63) 2. The language and sentence making of methods section need to be polished. Few sentences are not clearly expressing the performed steps. Response: Thank you for your kind suggestion. We have revised the methods section. 3. Why alphafold2 server was used, there are many other validated servers for structure prediction including Itasser, Trosetta etc. Justify the use of this specific server in this study? Response: Thank you for your kind suggestion. There are 113 amino acids in 2C12, its structure is not complicated, and we are familiar with alphafold2, which was also used in our previous study for protein structure prediction (26). After the structure prediction, further validation and quality assessment of the 3D structure were performed, and the predicted results were reasonable. 4. The 3D structure of 2C12 was predicted using alphafold2, but further validation and quality assessment of the 3D structure is missing. A proper validation which includes ERRAT quality score, favored region in Ramachandran plot and z score need to be provided with figure. Response: Thank you for your kind suggestion. We have provided description about validation and quality assessment of the 3D structure in the revised manuscript. (Line182-184) Further validation and quality assessment of the 3D structure were performed, the ERRAT quality score was 97.0588, the Ramachandran plot: 94.6% core, 5.4% allow, 0.0% gener, 0.0% disall, Z-Score: -6.08 (Supporting Information:Figure S1 and S2). The Z-score is plotted by the online version of the tool ProSa, which is not loaded. 5. Some of the explanation in discussion needs proper explanation. For example, this line “Molecular docking analysis using ZDock3.0.2 revealed that 2C12 and HPV16 L1 formed five pairs of hydrogen bonds as well as multiple hydrophobic interactions”, what is the significance of hydrogen bond and hydrophobic interaction. Appropriate justification with reference is vital. Response: Thank you for your kind suggestion. We have added the description about hydrogen bonds and multiple hydrophobic interactions in the revised manuscript. (line237-250 and line 293-297) 6. Compare some of the findings with previous study which is missing in the discussion. Is there any similar study or findings exist for AHPVD and AHPVT, compare with other relevant studies? Response: Thank you for your kind suggestion. We have added the discussion of previous studies (line263-269 and line270-274). 7. Illustrate figure 4 more clearly, you can improve the quality and resolution of the picture. Response: Thank you for your kind suggestion. We have revised the manuscript and Figure 4. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0311137.r002
10 Sep 2024 Decision Letter - Haitham Mohamed Amer, Editor PONE-D-24-26930R1Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16PLOS ONE Dear Dr. Bai, 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 Oct 25 2024 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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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: N/A ****** 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: Yes ****** 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: (No Response) Reviewer #2: (No Response) ****** 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: No Attachments Attachment Submitted filename: PLOS One Review_Round 2.docx https://doi.org/10.1371/journal.pone.0311137.r003
Revision 2
11 Sep 2024 Author Response 1. Line 48 – the term” physical therapy” has a very specific meaning, and their usage in this context is incorrect. The authors should state what clinical intervention for atypical hyperplasia/neoplasia they are referencing, such as Cryotherapy, Laser ablation, Loop electrosurgical excision procedure (LEEP). ...Cone biopsy, etc. Response: Thank you for your kind suggestion. We have revised this section. (line 46-49) 2. 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. Response: Thank you for your kind reminder. We have revised the reference 19 correctly. (line 382-384) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0311137.r004
16 Sep 2024 Decision Letter - Haitham Mohamed Amer, Editor Design and antiviral assessment of a panel of fusion proteins targeting human papillomavirus type 16 PONE-D-24-26930R2 Dear Dr. Bai, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Haitham Mohamed Amer, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311137.r005
Formally Accepted
15 Oct 2024 Acceptance Letter - Haitham Mohamed Amer, Editor PONE-D-24-26930R2 PLOS ONE Dear Dr. Bai, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Haitham Mohamed Amer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311137.r006

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