PONE-D-24-12287Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of AfricaPLOS ONE

Dear Dr. Balam,

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.

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PLOS ONE

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Reviewers' comments:

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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 manuscript reports essentially confirmatory data on the recognition of Pvs48/45 by antibodies in people previously exposed to P. falciparum thus confirming cross-reactivity on a larger sample size from 4 different African countries. The authors have cited previously published work on the same phenomenon of cross-reactivity. The work in the cited reference showed >70% cross recognition by sera from a study conducted in Southern African region (Zimbabwe). While the studies and data support the conclusions drawn, I would like to see authors respond to my major and minor concerns listed below:

1. Line 61:replace ...,"(exposed only to P. falciparum)".... with ....."exposed predominantly to P. falciparum"...., unless authors have tested blood for infecting parasites. Co-infections with other Plasmodium species are quite common.

2. Lines 83-97: should be deleted because they are irrelevant to the studies.

3. The Introduction is vey unfocussed and very similar superfluous and needs to be carefully made succinct with the overall goal of studies properly stated with due credit to previously published work.

4. 2.1 and 2.2 have very similar details and efforts need to be made to minimize duplication.

5. 2.3: Do not include results in the methods.

6. 2.4 sub heading can be deleted and details can simply be stated because they have been published previously and well known at this point.

7. Similarly 2.5 details are likely published in 62 and be referenced without the need for a sub-heading.

8. Line 77: what is the superscript 2 on the word derivatives for?

9. 2.6: Why have authors included Ref number for each chemical from a particular vendor?

10. This reviewer does not like the term competition term for the ELISA. All they are doing is to self deplete cross-reacting antibodies. In the same they include ref. 72. Are any details different from those in the indirect ELISA?

11. 2.9: Why did they include complement in the SMFA? Does it depend upon complement?

12. Line 361: the reference 81 pointed out above was from a study on sera from Africa and not Latin America and that study too showed >70% sera positive for cross-reactivity.

13. The studies on P. falciparum SMFA with affinity purified are nice and not surprising. What the authors must do is to evaluate the same antibody in DMFA with P. vivax. In the absence of this data the present studies are only confirmatory and add incremental knowledge.

14: Comments on Figures:

Figures 1,2 and 3: Delete Panel A and simply add % responded column in the table. In fact I would suggest that modified Figures 1-3 can simply be three panels of a single Figure.

Figure 4: It can be deleted because it does not add any knowledge. Panel A simply shows that BF40 has less antibody than BF 70. Panel B ELISA titration curves for a few select sera. Panel C just shows which eluted fraction has maximum eluted antibody amount. The entire figure does not contribute anything new.

Figure 5: The authors need to demonstrate IgM (day 270-280) and IgG (days >300) responses against Pfs48/45 after boosting with parasite lysates. The overall IFA quality was unacceptably poor.

Reviewer #2: This is an interesting report suggesting cross-species protection by a Plasmodium vivax antigenic protein possibly involved in transmission-blocking activity against P. falciparum.

There is one major concern with the interpretation offered. The authors confidently assert that the African sera examined would have never been exposed to any P. vivax malaria at all. This view seems to hinge on the sera being collected prior to the now well known and commonly reported presence of endemic P. vivax in all of the nations represented in their samples. This presumes the flurry of P. vivax reports out of Africa starting in about 2010 documented a new invasion of the continent and that prior to this, no P. vivax transmission occurred. This is very highly unlikely. Instead, P. vivax transmission has very probably always occurred in Africa but went unnoticed due to inherently very low parasitemias ,or due to microscopic misidentification as P. ovale. This reviewer does not accept the presumption of cross-reactive P. vivax antigen in people who very probably had experience with P. vivax infection.

The authors should consider the likelihood of the observed seropositivity being due to actual exposure to endemic P. vivax.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-12287R1Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of AfricaPLOS ONE

Dear Dr. Balam,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has been improved and has merit but it still 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 additional points raised during the review process, as outlined below.

Please submit your revised manuscript by Jan 27 2025 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

David Joseph Diemert, M.D.

Academic Editor

PLOS ONE

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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

**********

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: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: 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

**********

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

**********

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: It is obvious that the authors have revised the current manuscript extensively and it is much improved from the previous original submission. Basically the findings confirm previous similar results reported in Ref. 50, however the studies do provide demographically broader cross-reactivity. I suggest following comments for authors to take into consideration to help improve the overall presentation:

1. The Literature should remain focused on the target antigen and the intent should not be to review the literature on P. vivax vaccines. This will focus the manuscript and also help remove unnecessary too many self-citations.

2. Why are there are no co-authors who collected and provided sera from TZ and NIG?

3. line 53: Pf IFA data is questionable because the sera likely contained anti-Pf IgM induced by the Pf booster dose and it may not be due to boosted anti-Pvs48/45 abs.

4. Provide a more recent ref for #3 for WHO

5. Many references are irrelevant (in view of my point 1) . These include 17-19, 23, 25-27.

line 123: it should be plasma because the blood was collected in the presence of EDTA.

6. lines 137-146: I understand these mice data were published previously but the same mice mice after rest period were boosted with Pf. In that case lines 137-146 can be simply replaced with a sentence with appropriate reference.

7. line 165:. Change to "....The reaction was stopped using ...ul 1 M sulfuric acid and the optical density measured.... was the OD measured at both the wavelengths ?

8. Lines 178-183: If these beads were prepared as published then just say so and cite the ref. and delete these lines.

9. Line 253: You don't know whether the conformation was the same or not. You are only looking at the ag-ab reactivity.

10. In Figs. 1 and 2 when presenting human sera OD values - are these observed or after subtraction of (negative control = 3SD)? If these are after subtraction then I don't have any further comment. If not, then the right way will be to present net OD after subtraction. In the latter case all further % values and calculations throughout the manuscript are likely to be affected.

11. sub-heading on lines 274-274: Somehow the use of Concerning is not sitting well in my reading. The authors might want to rephrase.

12. Lines 317-322:Suggest rewrite "...., was then used in SMFA for functional cross-reactivity against Pf gametocytes (Table 1). At the end of this paragraph, the authors should state why they could not those abs against Pv in DMFA.

13. line 338: replace vigorous with strong.

14. ...260. Further immunization with a single dose of Pf gametocytes resulted in significant boosting of anti-Pvs48/45 ELISA....

15. Line 355-357: IFATR concern in view of my comment number number 3.

16. line 369: ....collection, confirms previously reported cross reactivity (ref. 50).

17. Line 375: Move ref. 50 to modified line 369 and ref. 51 is not from mice but humans.

18. Line 397: ...exposed repeatedly. and delete periods as a function of their age from line 398.

19. Line 432: I am not convinced about IFAT data against Pf,as stated before.

20. Line 434: ...in mice provide further support for Pvs48/45 as a target for TB vaccine.

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Reviewer #1: Yes:  Nirbhay Kumar

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[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.

PONE-D-24-12287R2Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with plasma from Plasmodium falciparum endemic areas of AfricaPLOS ONE

Dear Dr. Balam,

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 Mar 02 2025 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

David J. Diemert, M.D.

Academic Editor

PLOS ONE

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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.

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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

**********

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: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: 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

**********

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

**********

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: All my previous concerns have been addressed except one pertaining to IFA data. I would suggest that authors insert a disclaimer (on lines 340 and 426-427) that the IFA reactivity to Pf may also be due to primary IgM response against immunizing Pf gametoc4yte proteins (not tested).

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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: Yes:  Nirbhay Kumar

**********

[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.

Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with plasma from Plasmodium falciparum endemic areas of Africa

PONE-D-24-12287R3

Dear Dr. Balam,

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.

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David J. Diemert, M.D.

Academic Editor

PLOS ONE

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