PONE-D-24-29510Waning of anti-SARS-CoV-2 antibodies after the first wave of the COVID-19 pandemic in 2020: a 12-month-evaluation in three population-based European studiesPLOS ONE

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I have read the journal's policy and the authors of this manuscript have the following competing interests: 

A.W. and M.H. report personal fees and non-financial support from Roche Diagnostics. A.W. and M.H. report non-financial support from Euroimmun, non-financial support from Viramed, non-financial support from Mikrogen. A.W. and M.H. report grants, non-financial support and other from German Centre for Infection Research DZIF, grants and non-financial support from Government of Bavaria, non-financial support from BMW, non-financial support from Munich Police, nonfinancial support and other from Accenture. A.W. and M.H. report personal fees and nonfinancial support from Dr. Box-Betrobox, non-financial support from Dr. Becker MVZ during the conduct of the study. A.W. is involved in other different patents and companies not in relation to the serology of SARS-CoV-2. A.W. reports personal fees and others from Haeraeus Sensors, nonfinancial support from Bruker Daltonics, all of which are outside the submitted work, and non-related to SARS-CoV-2. The other authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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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

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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: No

Reviewer #2: Yes

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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:  The manuscript by Dr Novelli and colleagues aimed to study the evolution of IgG antibodies to SARS-CoV2 Spike antigen over time. To that end, the authors used three cohorts, from Germany, France and Luxemburg, established in the first half of the year 2020 in the three countries. For their study, they included a subpopulation in each of the three cohorts and tested selected plasma samples with an ELISA assay allowing the detection of IgG to SARS-CoV2 S protein.

After testing 65, 599 and 16 samples of the cohorts from Germany, France and Luxemburg, respectively, the authors observed no sero-negativation four months after infection and then a reversion in roughly 1/3 of the patients 6 and 12 months after infection. The authors concluded on the waning of IgG antibodies 6 to 12 months after primary infection in one third of infected persons in their cohorts.

The authors addressed here an important issue in the immunity to SARS-CoV2 and its duration. This type of study might also help address the question of herd immunity. The authors are commended for that.

The work presents also limitations, some of which mentioned by the authors in the discussion. These include:

1. The role of re-infection in the maintenance of IgG antibody level. This point is important and should be discussed further, especially in the light of what is known on covid-19 in the three countries during the study period.

2. Although the 3 countries are neighbors, political decisions during the early phase of covid-19 were not exactly the same and this might have some incidence on epidemiological situation in the three countries. This should be mentioned in the manuscript.

3. The initial cohorts from the three countries included large sample size. However, in the studied population, the sample size per site is very disequilibrated (16, 65 and 599). Did the authors compared these 3 populations? How comparable were they? If they were not comparable, then it is very risky to draw a general conclusion on antibody dynamics from 3 different population in size and structure.

4. Can the authors indicate the sensitivity and specificity of the ELISA assay used?

5. The authors defined two categories in the ELISA results according to OD ratios: below 0.7, the samples were considered negative and above 1.1, they were considered positive. What about those in between? It is written in the manuscript that samples with OD ration above 0.7 were tested for neutralization. How many of those with OD ratio between 0.7 and 1.1 were positive in neutralization (i.e. as per authors’ definition with a titer above 40)? This should be helpful to readers and should be presented.

Reviewer #2:  Waning of anti-SARS-CoV-2 antibodies after the first wave of the COVID-19 pandemic in 2020: a 12-month-evaluation in three population-based European studies

In this study, the decrease in antibody levels in unvaccinated Anti-S IgG seropositive individuals from three different centers in Europe was investigated as a retrospective cohort study. The inclusion of Anti-S IgG reactive individuals in the early phase of the COVID-19 Pandemic (in 2020) may be valuable in terms of understanding the effect of natural infection on antibody kinetics in the absence of the vaccine factor. It is appropriate to publish after the necessary major and minor revisions.

Major

1- The principled test used for the virus neutralization test (VNT) should be specified in the Material - Method section. In this context, PRNT (Plaque Reduction Neutralization Test) or Surrogate Neutralization Tests can be used [1].

• In addition, the diagnostic reference values of the neutralization test results should be specified according to the relevant method used (positive and negative limit values).

2- Has a power analysis been performed to support the sample power? If so, it should be stated in the Material-Method section.

3- The variant that was effective at the beginning of the COVID-19 pandemic was D614G. Later, the Alpha (B.1.1.7.) variant was effective. In this context, the development of reinfection in the cases included in the study should be taken into consideration and explained.

4- The reason why Anti-N (Nucleocapsid-specific antibody), which is an important antibody in the post-infection situation, is not preferred instead of Anti-S measurement after SARS-CoV-2 infection should be explained.

5- In the results of the study, it was stated that seroreversion was observed more frequently in the 30-39 age group compared to the 15-29 age group and that it was statistically significant (p<0.001). At this point, it was stated in the discussion section that the 30-39 age group is more socioactive as a possible reason underlying the more frequent seronegativity observed compared to the older age group. This explanation may be possible due to the possibility of early infection. However, in line with basic immunological phenomena, there is more antibody production in young individuals [2, 3]. For example, while 4000 IU/mL of Antibody can be formed in a young individual, 2000 IU/mL of antibody can be formed in an older individual. In this case, it is interesting that a young individual may have early infection but falls into seronegativity earlier or more frequently than an older individual within the elapsed time period. This situation should be clarified from an immunological perspective and further explanations should be provided.

6- As a result, seroreversion was investigated in the cases included in the study in line with decreasing antibodies. Although scientific inferences have been examined very well at this point, recommendations should also be shared in terms of public health. What is recommended for individuals who are seronegative within a certain period of time?

Minor

1- Virus neutralization test principle and commercial kit identification, if used, should be provided.

References

1- Dinç, H. Ö., Demirci, M., Özdemir, Y. E., Sirekbasan, S., Aktaş, A. N., Karaali, R., ... & Kocazeybek, B. (2022). Anti-SARS-CoV-2 IgG and neutralizing antibody levels in patients with past COVID-19 infection: a longitudinal study. Balkan Medical Journal, 39(3), 172.

2- Castellao Tavares, S. M. Q. M., Bravo Junior, W. L., & Alves Leite, J. L. (2015). Analyze the levels of immunoglobulins IgG and IgM in elderly and youngs. IntJ Immunol Immunother, 1 (1).

3- Mattos, M. S., Vandendriessche, S., Waisman, A., & Marques, P. E. (2024). The immunology of B-1 cells: from development to aging. Immunity & Aging, 21(1), 54.

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Reviewer #1: No

Reviewer #2: Yes:  Bekir Kocazeybek

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Waning of anti-SARS-CoV-2 antibodies after the first wave of the COVID-19 pandemic in 2020: a 12-month-evaluation in three population-based European studies

PONE-D-24-29510R1

Dear Dr. Novelli,

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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Kind regards,

Asli Suner Karakulah, PhD

Academic Editor

PLOS ONE

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

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: 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

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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: (No Response)

Reviewer #2: The authors described waning in anti-SARS-CoV-2 IgG in adult general populations infected during the first wave of the COVID-19 pandemic in 2020 across : From population-based cohorts, anti-S IgG levels remained stable during

the first 4 months following SARS-CoV-2 infection. They concluded that mMost of the decay occurred afterward; nearly one-third of people seroreverted 6 and 12 months later. Low IgG levels and seroneutralization negativity were independent predictors of seroreversion. All my comments are answered and applied to the text.

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Reviewer #1: No

Reviewer #2: Yes:  Bekir Kocazeybek

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