PONE-D-21-00516

Serological Profiles of Pan-CoV Responses in COVID-19 Patients Using a Multiplexed Electro-chemiluminescence-based Testing Platform

PLOS ONE

Dear Dr. Bergmann-Leitner,

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.

Both reviewer find the article of interest, even if many improvements are suggested by the reviewer 1, the results remained important even if a full description of the study limitations deserved to be added.

The main concern of the two reviewers, however, is the choice of the naïve population from a very different epidemiologic context: US population versus a Korean military group). This deserved to be adressed carefully and I suggest to add some data from a pre-epidemic Korean population or even from people previously exposed to other corona viruses as it is expected to be found in Korea.

Please submit your revised manuscript by Mar 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Pierre Roques, Ph.D.

Academic Editor

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: No

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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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: *Summary of the research

The clinical presentation of covid-19 (+) is wide, inconstant ad emerging, making the diagnostic, management and any implementation difficult. In response to that, the manuscript claims to value a new 10-plex electro-chemiluminescence assay as a multitasking Covid-19 screening platform. The dual IgM/IgG detection, respectively targeting Covid-19 Spike Covid-19 NP proteins, allows to achieved high specificity and sensitivity levels with a low samples-consuming. The cross reactivity is also considered in diagnosis to distinguish the covid-19 (+) persons (ill or cured) from the healthy population and/or covid-19 (-) persons and be a high-throughput sero-surveillance tool of quality.

*Overview of the manuscript’s

The multiplex approach is a commonly tool used in serological survey, including SARS-Cov2 as here : 1) The IgG/IgM detection allows to determine host responses profile independently to clinical signs and compared to control group, 2) The SARS-Cov-2 10-plex raises on the cross reactivity trouble. Nevertheless, this lacks of creativity " with a feeling of "déjà vu". The choice of a ECLIA approach remains unclear in implementation: diagnostic (combined with RT-qPR), management or survey (prevalence)?

Several papers already demonstrated the multiplex tools accuracy (%specificity and % sensitivity) to IgM and/or IgG detection in various population groups sera. Nevertheless, at the end of the discussion, the goal seems not to be fully achieved. Despite adequate statistical analysis, the data seems not enough exploited to draw the supported conclusions.

As reporting in others similar studies, the cross-reactivity concern between coronavirus is also discussed here. Its impact in the tools performance and the developing some plateforms depends on targeted population by covid-19. The sampling selection process and the choice of population categories are not enough justified. The groups detailed description lack and reducing the interpreted accurate. The authors forgot to address some possible limitations of the research such as a military environment choice: healthy and male person in majority.

To sum up, the improvement involves in giving details of the sampling collection process and providing more patients information. Analyses and discussion need to enrich in order to link the findings of this study to the supported conclusions (confer below recommendations).

*Recommended course of action (major)

1-Population study and selection criteria, and sampling:

-The choice of population groups and selection criteria support the study quality. The covid-19 and non-COVID-19 patients are enlisted at the same site and in the pandemic time period. However, the sampling process needs to be precise. The selection criteria has to be the same between these both populations in order to perform comparative study.

-Moreover, the negative status of non-Covid-19 patient (working in health center) seems only based on the observation (clincal forms) (evaluation bias) and/or the paitent declaration during the interview (no risk of exposition?) (= memory bias). While the prevalence of COVID-19 in Health Care Worker is low, providing evidence of (RT-qPCR) tests for non-covid-19 group is recommended.

-The military (covid-19 group) as well as the health care persons (non covid-19 group) are often considering as healthy and volunteer persons in participating in study compared to the general population. This behavior may impact on serological results and needs to be taking into consideration (analyses and discussion).

-The selection of pre-pandemic samples from several sources in the United States in 2019 is also questioned (Random or voluntary strategies?). In addition, epidemic information about coronavirus circulation during this 2019 summer would allow to determine whether the exposition to others CoV species differs between pre-pandemic samples and pandemic samples (Covid-19 (+) and Non-Covid-19 (-)) and enrich the cross-reactivity analyses.

-The lack of homogeneity in the inclusion criteria between 3 groups making difficult the value of antibody responses and their interpretation.

2-Sex and ages:

As influencing the antibodies response (i.e.= kinetic, duration, level), the sex and ages need to be noticed in groups.

3-Results graphic:

In addition to table 1, the illustration of patient features through a timeline-scale would better link the diseases progression, onset with the antibody responses and potentially other determinants factors.

*Recommended course of action (minor)

1-Severity level determination:

The illness definition and the levels severity remain uncleared for the covid-19 group: is it related to a score scale and performed by a same medical person (table 1: page 186)?

2-Chapter reorganisation:

The 334-347 discussion paraph, including particular objectives, should be moved in the introduction than in the conclusion to facilitate the results analyses and authors goals.

-Results presentation in only 2 major parts "IgM/IgG characterization" and "the cross reactivity events" might facilitate and open the discussion and subchapters could be suggested.

*Specific areas for improvement in discussion

1-Onset diseases importance or delay in clinical signs apparition:

-The ELCIA approach allows to characterize the COVID-19 samples compared to pre-pandemic and control based on IgM/IgG detection with a high sensibility (100- to 200-fold higher).

The authors claim that by using a larger data set, ELCIA would have high potential for predicting acute infection status and exposure of an individual from their serological data. If is it true: how to explain the difference between id-003 and id-009? Indeed, the id-0003 and i-0009 subjects showed the same mild clinical presentation and the timeline of samples collection.

As arguing the authors, the absence of antibodies response to Subject i-0003 may be related to mild clinical presentation mild symptoms and/or the delay of serconversion (only one day following initial test positivity). If so, what about the interpretation of the id-009 results, showing similar clinical profile as the id-0003 patient (215-218)?

-As mentioned above, the authors suggests that the delay in antibodies response is related to the low clinical symptom: How explaining the IgM and IgG profiles of the id-007, id-008, id-0010? = asymptomatic patients whose samples, collected + 2 days after PCR (+), are associated to significant antibodies response?

-To argue in the IgM/IgG conjugated detection importance in Covid-19 management and/or survey, discussion should be enriched with additional related references.

2-Cross-reactivity:

The authors address possible limitations of the research, including cross-reactivity

To gain insights into the pre-existing immunity Covid induced antibody responses impact, the distinct patterns of cross-reactivity in IgM and IgG responses are plenty explored in the literature, such as the multiplex tools use, as here. The cross reactivity appears higher with IgG SARS-CoV-2 than the IgM SARS-CoV-2 binding antibodies. The author provided a long analysis in the cross reactivity trouble between coronavirus species. SARS-CoV-2 IgG antibodies may be cross-reactive with SARS-CoV-1 and MERS-CoV: 1) 33% of CoV-2-seropositive COVID-19 samples were also seropositive for MERS-CoV, 2) 50% IgM seropositivity for MERS-CoV was detected the Control Group (275-277).

In view of the % in MERS/SARS COV-2 cross reactivity, the MERS serological status of control population should be clarify : Is it a lack in specificity of the ECLIA approach or a real high MERS prevalence in control populations?

3-Confunding factors?:

As mentioning in the results revision, details about patients (sex and age) may help authors as well as reviewers by arguing some exceptions instead of criticizing the tools accuracy.

The cross relationship between antibodies profile and clinical presentation needs to be discussed according to some host determinants. The sampling design impacts also in the way.

-Example concerning sex determinant: the proportion of female is lower in the military population compared to general population

-Example concerning military group: this population is better physical fitness, influencing the host responses to pathogen.

-Example concerning control group: even not being directly in contact to Covid (+) patients, people working in hospital have usually better style of life than the general population (or take better care of their health).

All of these justify the above revision request by giving details about the study of population and arguing on the criteria selection etc...

*To go to further…

By combing IgG and IgM results, the authors success in mapping the course of this subject’s antibody response from the pre-COVID-19 or non-infected region to the COVID-19 region independently to clinical symptoms. This view may help in vaccine implementation as well as vaccine efficacy. Nevertheless, this is not fully achieved in view of the id-003 antibody response (low antibody response, low duration or delay?). The results of serological profile of id-0003 need to be checked with others tools (ELISA commercial kit or ELISA in house) to conclude on the ELCIA tool performance/limitation.

The id-003 data calls to future research on IgA profile and/or seroneutalization. It should be important to mention that the diseases progression depends on the seroneutralization antibodies pool as well as the IgA kinetics (i.e. IgA secretion in local).

Unfortunately, the authors don’t open up enough perspectives.

*Current cutting-edge tool: is it? and for what?

The authors could bring more explanations in why: 1) this new multiplex coronavirus antigen evaluation may change the covid-19 survey; 2) the electro-chemiluminescence assay platform (serological high-throughput testing of sera/plasma) may support the current molecular covid-19 diagnosis?

*Additional minor points:

1-Rewrite the abstract

Shorter by highlighting the goal and the achievement or not of the study

2-Additional references

In introduction, the authors explain in why the study matters and put the research in context: antibodies response (kinetics of apparition, the delay in apparition, the lack of duration).

But they don’t precise in why ECLIA approach may be revolutionary compared to others?

Literature lacking in other serological approaches (ELISA gold standard, etc...) as well as about the comparative studies between various serological tools (ELISA vs ECLIA).

*Keys list of the paper

-Strengths:

Ethical guidelines respect

Exhaustive protocol description

Adequate statistical analyses

Graphics support the findings

-Weaknesses:

No creative concept or idea, “déjà vu concept”

Absence in population samples information and unclear election criteria

Scant or incomplete data explanation to draw conclusion

Non results synthesize prior discussion

Reviewer #2: Reviewing report for PLoS One PONE-D-21-00516 : “Serological Profiles of Pan-CoV Responses in COVID-19 Patients Using a Multiplexed Electro-chemiluminescence-based Testing Platform” from S. Chaudhury et alii.

In this report, S Chaudhury and coworkers exposed the outcome of a multiplexed electro-chemiluminescence-based assay (ECLIA) detecting pan-coronavirus immune response in the context of the COVID-19 pandemic. The assay has the capacity to detect IgM ad IgG response against full-length spike of various coronavirus and epitope specificity of the response against spike protein subdomains including receptor bind domain (RBD).

The study was conducted with COVID-19 patients and controls recruited in Korea while a third group of pre-pandemics patients was recruited in the US. Each group was composed of 10 patients. These rather small size is compensated by the depth of data analysis performed by the authors.

An important outcome of this study was that authors did not observed any association between seropositivity and disease severity.

The authors observed that in favorable circumstances, the ECLIA test is very sensitive allowing a dilution of up to 30.000. This high sensitivity was however incapable to detect a specific immune response in a COVID-19 patients, sampled 24H after initial diagnosis. This patient was virtually useless in the whole study (except to remind that serology should not be performed too early post disease onset).

The ECLIA revealed a substantial cross-reactivity of IgG between SARS-CoV-2 and various other coronaviruses either epidemic (SARS-CoV-1, MERS-CoV) or endemic (HKU1 and OC43). The differences between IgM and IgG specificity explain why the authors suggest that a machine-learning approach using a large set of data should be used to correctly diagnose a recent or acute exposure to SARS-CoV-2 (line 424). This complexity suggests that the interpretation of ECLIA is plausibly possible only for a given geographical context (ie exposed to a broadly common infectious environment) using controls coming from the same place than putative patients to be diagnosed. This aspect is not really developed by the authors. I suggest that they add a comment in the final version about the origin of these pre-pandemic plasmas as a potential limitation of their work (South Korean pre-COVID might have yielded an IgG cloud on figure 3 much closer or even overlapping with the COVID-19 cloud).

Overall, in the context of COVID-19 pandemics, the work is interesting and bring valuable insight about the various components building the immune response against SARS-CoV-2. It emphasizes as well the technical versatility of ECLIA (linear range, scalability, etc…).

The reader might regret that pre-pandemic samples have been collected from subjects living in the US, a country that, at variance with South Korea, was not as affected by previous SARS-CoV-1 and MERS-CoV epidemics. However this situation does not seem to alter the overall conclusions of the paper despite the absence of any long-term seroreactivity to MERS-CoV or SARS-CoV-1 in pre-pandemic plasmas

Minor issue;

Line 216: “his plasma samples” not “their”

Line 364: “ECLIA”, “must be validated”

Figure 3: IgG seropositivity, the smallest-greenish piece of the pie represents 10% not 20%.

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Reviewer #1: No

Reviewer #2: Yes: Pascal Pineau

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PONE-D-21-00516R1

Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform

PLOS ONE

Dear Dr. Bergmann-Leitner,

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.

The two independent reviewers raised the completely opposite comments: accept and reject. Thus, I asked the third reviewer to give us the new comments, and it was accept. So please make a rebuttal against the comments provided from the reviewer 1. I will give a decision for the publication.

Please submit your revised manuscript by Jun 24 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Etsuro Ito

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.

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: The authors made efforts to answer to any reviewed points.

1-Concerning the first point :

Although not being a well-design randomized trial, the recruitment process has to be described and is always missing here.

Above all, it needs to be the same between populations groups (example of additional required information: inclusion, exclusion criteria etc..).

The small size of sample is the major limitation of study. The authors are aware and, unfortunately, it can’t be solved at this point of revision.

Determining the feasibility of the multiplex ECLIA assay is based on deep and multi-variable statistical analysis, which being impaired by this minimal number of person (see above) as wel as from a unrandomized population selection (‘convenience’ samples)

In addition to the heterogenicity of the sample groups, it is still not precised if the developped method interest is to the diagnostic (1) and/or the surveillance (2).

(1) In view of the current epidemiological context, diagnostic tools need to be operational to respond to emergency (outbreak), opposing the authors sentence "a rigorous case-control study was infeasible in the midst of an emergency outbreak response".

(2) In addition, a sero-surveillance tool development in "real-world" needs to be reliable, in continue, meaning to be evaluate from disease progression data. Both unfeasible in emergency context and unvalued in accordance to the illness outcome, the interest of this tool is questioned.

It is even less useful to the preparedness as authors saying being a " pre-pandemic to pandemic imperfectly comparison study. "

To conclude, the first answer remains unsatisfactory.

2-The second point is partially it. While the authors claims that the control samples were tested for COVID-19 using the same diagnostic assay as the COVID-19 patients, the "RT-PCR or RT-qPCR" molecular approach is not precised.

3- The accuracy of a clinical study is based on precise and available personal information of participations and not on approximative data " age range and sex for COVID-19 and Control subjects in Table 1 and in S1 Table". Unfortunately, the militaries, the U.S. Department of Defense civilians and contractors are far from to reflect a wide demographic range of U.S. population, including manual worker and/or low-class.

4-The fact that it is not possible to determine general trends in pre-pandemic coronavirus exposure in these individuals with diverse geographic history makes antibody responses analyses inaccuracy (specificity, sensitivity, cross specificity, immune response memory)

5-The ages have not to be approximative in the groups.

6- This point is satisfied by additional sentence

Minor points

The authors took the trouble to answer at any minor points although being aware of any study limitations: an un sufficient power to identify significant differences in serological data with respect to sex and age, a high (apparent 50%) IgM seropositivity for MERS-CoV in the Control Group and absence of diseases progression. These three points oppose to validation this tool use in the Covid-19 management and/or survey.

As being much more sensitive than a regular ELISA (we had previously published formal, extensive comparisons of the platforms), ECLIA may be attractive in the screening campaign by in particular conjugating the IgM/IgA than IgM/IgG.

Conclusion

The authors’ efforts in rewriting, in providing evidence and in arguing reviewer comments are visible. In view of preliminary results, enrich works by increasing the size of population groups, by providing the personal information and by following the diseases progression or conversely, the asymptomatic forms maintenance should be carried out.

From that, a sufficient accuracy is hoped to reach as well as to erase the study limitations and to convince to develop the platform of covid diagnostic based on ECLIA process. But, It is not the case now.

Reviewer #2: (No Response)

Reviewer #3: The manuscript entitled “Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform” by Prof. Elke Bergmann-Leitner presented a new 10-plex electro-chemiluminescence-based assay to measure IgM and IgG responses to the spike proteins from multiple human coronaviruses including SARS-CoV-2, assess the epitope specificity of the SARS-CoV-2 antibody response against full-length spike protein, receptor-binding domain and N-terminal domain of the spike protein, and the nucleocapsid protein. The article has been carefully revised according to the reviewers’ comments and well organized. Publication in PLOS ONE is recommended after correction of some typos and grammar mistakes.

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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: Pascal Pineau

Reviewer #3: No

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.

Serological profiles of pan-coronavirus-specific responses in COVID-19 patients using a multiplexed electro-chemiluminescence-based testing platform

PONE-D-21-00516R2

Dear Dr. Bergmann-Leitner,

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Kind regards,

Etsuro Ito

Academic Editor

PLOS ONE