Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene

Silvanos Opanda; Wallace Bulimo; George Gachara; Christopher Ekuttan; Evans Amukoye

doi:10.1371/journal.pone.0228029

Peer Review History

Original SubmissionOctober 23, 2019
12 Nov 2019 Decision Letter - Ronald Dijkman, Editor PONE-D-19-29545 Assessing Antigenic Drift and Phylogeny of Influenza A (H1N1) pdm09 Virus in Kenya the Using HA1 sub-unit of the Hemagglutinin gene. PLOS ONE Dear Mr Opanda, 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. We would appreciate receiving your revised manuscript by Dec 27 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please amend the manuscript submission data (via Edit Submission) to include authors Wallace Bulimo, George Gachara, Christopher Ekuttan and Evans Amukoye. Additional Editor Comments: Dear Author, Both reviewers and myself, highly recommend you to include HAI antigenic data in the revised version to complement the current in silico data. Thank you for your time and understanding. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: 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: In their manuscript, Opanda et. al. describe an in silico study of A/H1N1 viruses circulating in Kenya from 2015-2018. The paper is well written, clear, and the data is important in showing the natural drift in influenza A/H1N1 viruses in Kenya and issues that might arrive from decreased vaccine effectiveness. However, to increase the novelty of the manuscript, more work should be done with data available from other sources as well as in vitro analysis to confirm these in silico results. A few major comments for improvement exist. 1) It is unclear if the strains used in this analysis are new, or are what is already available on the Influenza Research database (fludb.org) and on GISAID (gisaid.org)…if these are new strains, then the 46 available H1N1 strains from Kenya on IRD with addition of non-duplicates from the 121 strains available on GISAID should be included in this study as it will greatly increase the power of the analyses performed. This will alleviate the first limitation of the study, somewhat. 2) Ideally, it would be good to show HAI antigenic data (and even microneutralization data using reference serum) to show how these in silico results correlate to wet laboratory results. Indeed, antigenic cartography of these viruses would be a good way to further show drift and possible vaccine escape. This will definitely alleviate the second shortcoming of the study as mentioned in the discussion. 3) Lines 241-244: This is extremely important information and should be highlighted more in the results, as it is not discussed previously. 4) While calculating the predicted efficacy of the vaccine is useful (under ideal conditions, of course) ,it would be very good to compare the results from this study with the vaccine effectiveness calculated in Kenya for each year so as to show that these predictive values d hold true to their intended results. 5) How does this predicted drift compare to viruses isolated in the greater geographic area? Is there some hypothesis as to why the viruses in Kenya appear to be drifting in such a manner? Reviewer #2: Overall, the manuscript is well written the main points are clearly stated. The use of bibliography is adequate and the tables and figures are sufficiently clear to help interpret the results. Comments: -line 45: The first pandemic of the 21st century was SARS, please amend. -line 102: what primers? How large is the resulting fragment? Does it cover the complete ORF? -line 111: M13 sequencing primers: where is the description of the cloning vector and process used to sequence with the M13 primers? -line 123: What inference method was used? Also, how many generations where run and sampled every how many times. Any % burn-ins excluded? It is also important, going back to line 102, how large the gene fragments are to asses how reliable the analysis is. -line 125: please provide accession numbers in supplemental material -line 142: I would indicate that you should also consider the NGlyc result in your estimation of glycosylated sites. The algorithm is very specific in that a >/= score of (++) should be used for describing an N-glycosylation site with high specificity. I would include that criteria as well and only inform those sites that also meet that criteria. A bit more conservative, since you are using only computational analyses throughout this manuscript. -line 247: It is a good practice to state the shortcomings of a manuscript, however, you should also give a reason why. In this case it would have been very interesting to see how the HAI results correlate to the results of this manuscript. HAI is a relatively simple procedure if you have the reagents. At least give an explanation of why it was not incorporated. ****** 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 to be viewed.] 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. 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Revision 1
27 Dec 2019 Author Response Below is a point to point response to the comments by the academic editor reviewers: ACADEMIC EDITOR PLOS ONE: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming 2. Please amend the manuscript submission data (via Edit Submission) to include authors Wallace Bulimo, George Gachara, Christopher Ekuttan and Evans Amukoye. 3. Both reviewers and myself, highly recommend you to include HAI antigenic data in the revised version to complement the current in silico data. Thank you, all these have been addressed. REVIEWER # 1: In their manuscript, Opanda et. al. describe an in silico study of A/H1N1 viruses circulating in Kenya from 2015-2018. The paper is well written, clear, and the data is important in showing the natural drift in influenza A/H1N1 viruses in Kenya and issues that might arrive from decreased vaccine effectiveness. However, to increase the novelty of the manuscript, more work should be done with data available from other sources as well as in vitro analysis to confirm these in silico results. A few major comments for improvement exist. 1. It is unclear if the strains used in this analysis are new, or are what is already available on the Influenza Research database (fludb.org) and on GISAID (gisaid.org)…if these are new strains, then the 46 available H1N1 strains from Kenya on IRD with addition of non-duplicates from the 121 strains available on GISAID should be included in this study as it will greatly increase the power of the analyses performed. This will alleviate the first limitation of the study, somewhat. Thank you for this observation. The Kenyan A/H1N1 pdm09 HA gene sequence data used in this study is not new but are the ones already available in the genetic databases under accession numbers: ANH22064 - ARK18942; MH316121, MG815810; MH356637 - MH356647 and MK692755 - MK692776 in the manuscript (lines 119 – 120). The study focused on the sequence data generated during 2015 to 2018 influenza seasons in Kenya. 2. Ideally, it would be good to show HAI antigenic data (and even microneutralization data using reference serum) to show how these in silico results correlate to wet laboratory results. Indeed, antigenic cartography of these viruses would be a good way to further show drift and possible vaccine escape. This will definitely alleviate the second shortcoming of the study as mentioned in the discussion. Thank you for this suggestion. The HAI assay antigenic data has been incorporated in the revised manuscript to validate the in-silico analyses to alleviate the second limitation of the study (Table 3, lines 250 – 253). 3. Lines 241-244: This is extremely important information and should be highlighted more in the results, as it is not discussed previously. We concur with the reviewer. A statement highlighting the difference in vaccine efficacies between A/Michigan/45/2015 and A/Brisbane/02/2018 vaccine strains against Kenyan 2018 A/H1N1 pdm09 isolates has been incorporated in the revised manuscript results section (lines 231 – 235). Unfortunately, we could to provide HAI assay antigenic data between the Kenyan 2018 isolates and A/Brisbane/02/2018 reference anti-serum, since our lab ceased being recognized as an NIC in 2018 and therefore we no longer receive HAI assay Kits from WHO CC reference labs. 4. While calculating the predicted efficacy of the vaccine is useful (under ideal conditions, of course), it would be very good to compare the results from this study with the vaccine effectiveness calculated in Kenya for each year so as to show that these predictive values hold true to their intended results. Thank you for your suggestion. The calculated vaccine effectiveness across the study period (2015-2018) have been presented in the manuscript in lines 221-235 and summarized in Table 2. However, we would like to point out that no vaccine effectiveness studies involving clinical trials are ever done in Kenya due to cost considerations. Hence, we are unable to compare the calculated vaccine effectiveness against predicted/observed (via clinical trials) vaccine effectiveness for Kenya. 5. How does this predicted drift compare to viruses isolated in the greater geographic area? Is there some hypothesis as to why the viruses in Kenya appear to be drifting in such a manner? Actually, the drifting the A/H1N1 pdm09 strains reported here is not unique to Kenyan viruses. The bulk of amino acid changes observed affecting antigenic sites of the HA1 domain among the Kenyan isolates have also been noticed in the sequences of foreign strains included in the study. To address this question, additional supporting information S1 Fig, S2 Fig, S3 Fig and S4 Fig has been added in the revised manuscript. REVIEWER # 2: 1. - Line 45: The first pandemic of the 21st century was SARS, please amend. Thank you for this observation. This has been amended in the revised manuscript (line 45). 2. - Line 102: what primers? How large is the resulting fragment? Does it cover the complete ORF? These are A/H1N1 pdm09 HA segment-specific primers tagged with flanking universal M13 primers to facilitate ease of sequencing. We have included a citation in the revised manuscript to address this (line101). The HA segment was amplified using two sets of primers to yield two overlapping PCR fragments: frag 1: ⁓ 976 bp and frag 2: ⁓ 890 bp. The contig of the two fragments (1.8 kb) covers the complete open reading frame of the HA gene (lines 106 – 107). 3. -line 111: M13 sequencing primers: where is the description of the cloning vector and process used to sequence with the M13 primers? We did not clone the amplicons into a vector. Instead the M13 primer tags were incorporated to provide anchors flanking the amplicons to facilitate sequencing of the amplicons. The statement has been edited accordingly in the revised version of the manuscript providing the source detailing the process of sequencing the PCR amplicons with the M13 primers (lines 111-112). 4. -line 123: What inference method was used? Also, how many generations were run and sampled every how many times. Any % burn-ins excluded? It is also important, going back to line 102, how large the gene fragments are to assess how reliable the analysis is. Thank you for this comment. The phylogenetic tree was constructed using Bayesian Monte Carlo Markov Chain (MCMC) inference method. The MCMC was run for 10 million generations, with sampling every 1000 generations and a 10% burn-in. These statements have been incorporated in the revised manuscript (lines 124 – 128). 5. -line 125: please provide accession numbers in supplemental material. Thank you for your suggestion. Accession numbers of HA sequences of all reference strains included in the analysis has been provided as supplemental material (S1 Table). 6. -line 142: I would indicate that you should also consider the NGlyc result in your estimation of glycosylated sites. The algorithm is very specific in that a >/= score of (++) should be used for describing an N-glycosylation site with high specificity. I would include that criteria as well and only inform those sites that also meet that criteria. A bit more conservative, since you are using only computational analyses throughout this manuscript. We concur with the reviewer’s comment. A score of (++) describes an N-glycosylation with high specificity. However, as stated in line 146 of the manuscript we considered as score cut-off value of > 0.5 suggestive of an N-glycosylation. We took (+) and (++) scores as evidence of potential glycosylation. 7. -line 247: It is a good practice to state the shortcomings of a manuscript, however, you should also give a reason why. In this case it would have been very interesting to see how the HAI results correlate to the results of this manuscript. HAI is a relatively simple procedure if you have the reagents. At least give an explanation of why it was not incorporated. Again, we appreciate the reviewer’s comment. We have not only mentioned the shortcomings but also addressed the strengths of the study. The HAI assay antigenic data has been incorporated in the revised manuscript (lines 251 – 254) and they corroborate the PEpitope findings which is reassuring about the power of PEpitope model. Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0228029.r002
7 Jan 2020 Decision Letter - Ronald Dijkman, Editor Assessing Antigenic Drift and Phylogeny of Influenza A (H1N1) pdm09 Virus in Kenya Using HA1 sub-unit of the Hemagglutinin gene. PONE-D-19-29545R1 Dear Dr. Opanda, All the best wishes for 2020. We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Ronald Dijkman, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): 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: 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 https://doi.org/10.1371/journal.pone.0228029.r003
Formally Accepted
3 Feb 2020 Acceptance Letter - Ronald Dijkman, Editor PONE-D-19-29545R1 Assessing Antigenic Drift and Phylogeny of Influenza A (H1N1) pdm09 Virus in Kenya Using HA1 sub-unit of the Hemagglutinin gene. Dear Dr. Opanda: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ronald Dijkman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228029.r004

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