PONE-D-21-09100

Seroprevalence of SARS-CoV-2 Infection in Cincinnati Ohio USA from August to December 2020

PLOS ONE

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 Davis et al depicting the SAR-COv2 seroprevalence in Cincinnati Ohio USA for the time period August to December 2020 emphasizes on the rates of infection as well as serum IgG antibody for the viral Spike protein. ELISA seems more sensitive tool than Luminex for S-protein detection. They also monitored the average seropositive of S-antibody over the period of study to determine the loss of antibody. The age specific antibody production shows correlation between age groups and nullifies the fact of gender or ethnicity biasness. Their study showed that there is increase rate of infection in age group of below 30 as well as in later half of study which can be extrapolated to higher number for March 2021. This study also identified the geographical distribution of Cincinnati.

Overall, this study might help in National immunization program with a focus on development of herd immunity to the nation. I have few concerns that needs to be addressed:

1. Line 133/134, word “washed” missing.

2. The authors purified S and RBD domain ectopically expressing them in mammalian cell line. They have showed that 55.61% of S protein positive donors also positive for RBD domain. They did not clearly mention the reason of performing RBD positive donors. Most of the experiment that they performed were based on S protein only. This needs to be elaborated in discussion section.

3. Amongst the 108 donors who donated at least 2 times for this study showed 38 lost the antibody in second donation. It would be of more help for vaccination program to define the age group who lost S-protein antibody or whether it is some random phenomenon. Also same argument hold for 24 donors who showed positive for throughout the study.

4. The authors splitted for the study in two halves for better understanding of percent positivity of S-protein in 802 samples. What is the percentage positivity of Cov2 infection from samples collected for these two halves across Cincinnati or GCMA area?

5. The authors found some variation in S-protein positive cases as per geographic locations. Although the geographic variation is very little, a brief discussion might help readers to understand this variation within same state.

6. Line 314 - “March 2020” needs to be corrected to “March 2021”.

7. The National statistics by CDC shows that the highest rate of infection is almost 50% for White, Non-Hispanic followed by Hispanic population. The rate further drops to 11% for African- American and Asian had only3.3% of infectivity rate. How the authors can justify this variation in their samples in GCMA/Cincinnati area with respect to National infection rate?

8. Line 330 – Authors suggest that one vaccine dosage can give full protection with previously infected individuals. Given the data of loss of seropositivity in 38 donors of 108 for S-protein antibody on second test, authors needs to justify if this claim is safe overall by not giving two shots to them. Those individuals might not have the same level of antibody titer after having just one shot and might be of risk for potential infection instead of vaccination program which might put them in risk.

Reviewer #2: This study investigates the seroprevalence of SARS-CoV-2 infection amongst ~10K adult volunteers in the Cincinnati, USA by measuring serum IgG levels against the Spike protein (S). This is also compared to the serum IgG levels against the RBD protein. However, there seems to be no clear concordance between the two, which is alluded to the different sensitivity of the two ELISAs based on antigen size.

Please address the following comments:

1. Line 58 - The abstract should mention the exact timeline of the study, either 13th August to 8th December 2020, or mid August to first week of Dec, etc.

2. Line 99- please elaborate on how 'viral cellular involution' happens?

3. Line 112- it is not clear from this statement that the other HCoV strains were used to check cross-reactivity

4. Line 214- What was the OD cutoff for selecting the highly positive donors?

5. Line 216- What is the basis of 100% correlation? p-values would be useful here.

6. Line 308- reference?

7. Figure 1- Label X axis as OD units

8. Figure 2- Mention days/dates here. Or normalize the percent positivity by the no. of days for each month (especially for August and December)

9. Supplementary Figure 1E- Label the monomeric peak

10. Overall comment: what are the OD values in terms of antibody titers?

11. Were serum IgM levels against SARS-CoV-2 measured in these individuals?

Reviewer #3: Authors of the manuscript “Seroprevalence of SARS-CoV-2 Infection in Cincinnati Ohio USA from August to December 2020” have investigated rates of SARS-CoV-2 infection in 9,550 adult donors of the greater Cincinnati, Ohio, USA metropolitan area during the period of August to December 2020, just prior to initiation of the national vaccination program. They found overall positive prevalence of 8.40% for serum IgG antibody against the SARS-CoV-2 Spike protein. They also reported that males and females showed similar rates of past infection, and rates among different ethnic groups were not significantly different. They also reported lowest past infection rate among patients above the age of 60. Geographic analysis showed the lowest rates in the adjoining region of Kentucky (across the Ohio river). These findings about regional seroprevalence will help inform efforts to achieve herd immunity in conjunction with the national vaccination campaign. I would like to draw the attention of authors towards few points:

1. Line number 125/126 Board (IRB) reviewed the proposed SARS-CoV-2 serology initiative and classified it as non-human rearch. Please correct the sentence

2. The authors talked about significant variation in geographical analysis for past infection. Was comparison age matched as they observed age plays significant role so whether or not study population were matched for their age is important when considering geographical variations.

3. Authors should also discuss other studies with results showing variations in ethnic groups and should discuss their finding in comparison to CDC reports. They should discussion these studies too, if any done in the USA.

4. Detail statistical analysis for Table 4 will be helpful and a more explanatory table footnote will make it more clear to readers.

5. For figure 1.B p value must be indicated on the histogram

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Reviewer #1: Yes: Rupkatha Mukhopadhyay

Reviewer #2: No

Reviewer #3: No

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Seroprevalence of SARS-CoV-2 Infection in Cincinnati Ohio USA from August to December 2020

PONE-D-21-09100R1

Dear Dr. Molkentin,

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.

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Kind regards,

Binod Kumar, 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

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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: 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

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: (No Response)

Reviewer #2: Thanks for addressing the comments. The manuscript is acceptable for publication in the present format.

Reviewer #3: Authors have addressed all queries and answered them satisfactorily. The research article can be accepted for the publication

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Reviewer #1: Yes: Rupkatha Mukhopadhyay

Reviewer #2: No

Reviewer #3: No