Spike Antibody Titers Evaluation after a 2-Dose Regimen of BNT162b2 Vaccination in Healthcare Workers Previously Infected with SARS-CoV-2

The 2-dose BNT162b2 vaccine (Pfizer-BioNTech; reported efficacy 94.8%) regimen against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was authorized in December 2020 (1). However, studies suggest that previously infected individuals can achieve a rapid immune response with a single vaccine dose, compared with SARS-CoV-2-naive individuals (2–5). To test the effects of one and two vaccinations for individuals with a history of coronavirus disease (COVID-19), volunteers were recruited from among the medical staff at two local hospitals. All participants received two doses of BNT162b2 vaccine with a 3-week interval between doses. Blood samples were taken at days 27 to 0 (baseline), 2 weeks, and 2 months after the first vaccination. A quantitative determination of antibodies against the receptor binding domain of the SARS-CoV-2 S1 subunit of the spike protein was made using plasma samples (Elecsys anti-SARS-CoV-2 S, Roche Diagnostics International Ltd., Rotkreuz, Switzerland). Written informed consent was obtained from participants, and the study was approved by each hospital’s ethics committee. Statistical analysis was performed with EZR (6). Differences in antibody titers were evaluated by a t test. A P value of ,0.05 was considered significant. Overall, 369 health care workers were enrolled; 22 previously had COVID-19—confirmed by reverse transcriptase PCR (RT-PCR) during the past 3 months—while 347 did not have COVID-19 previously. To minimize confounding, the same number of noninfected individuals (COVID2) were randomly selected from among antibodynegative individuals at baseline to match the age and gender of each infected individual (COVID1). Chronological changes in antibody titers were compared between COVID2 and COVID1 groups (Fig. 1). Before vaccination, the baseline antibody titer was 213.6 (139.2 to 288.0) U/mL in the COVID1 group; the COVID2 group had no detectable antibodies. Antibody titers increased at 2 weeks in both groups; titers were significantly higher in the COVID1 group than in the COVID2 group (11,664 [9,155 to 14,174] U/mL versus 81.8 [21.2 to 142.2] U/mL; P , 0.001). However, the titers in the COVID2 group at 2 weeks did not reach the baseline levels of the COVID1 group (P , 0.001). At 2 months, antibody titers elevated further in the COVID2 group (P , 0.001) but not in the COVID1 group, although the titer remained higher than that of the COVID2 group (P , 0.001). No participant developed apparent COVID-19 during the blood sampling period. Two BNT162b2 vaccination doses were administered within 3 months of COVID-19 outbreak (between December 2020 and February 2021). We found that vaccination increased the antibody titers in SARS-CoV-2-naive individuals to values greater than the baseline levels in COVID1 participants. A further booster effect was not observed Editor Yongjun Sui, National Institutes of Health Copyright © 2021 Kayukawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Satoshi Kayukawa, kayu_kawa@yahoo.co.jp. Published 10 November 2021

In the manuscript, the authors analyzed the titers of Receptor binding domain of SARS-CoV-2 Spike protein-directed antibody in people with a history of COVID-19 or SARS-CoV-2-naive individuals after one and two doses of BNT162b2 vaccine. Based on the data obtained from a small size of samples, the author found that a two doses of BNT162b2 vaccine increased the antibody titers in SARS-CoV-2-naive individuals, while the second vaccination did not significantly increase the antibody titers in individuals with a history of COVID-19.The author's finding is supported by their clear data, although the data was obtained from a small size of samples as also discussed by the authors. The experiment and methodology of the manuscript are technically sound. The manuscript is suggested to be accepted. Specific comments are below. 1. The phrase "To verify that two vaccinations......" in line 23 should be changed to "To test the effects of one and two vaccinations......" to match the context of the manuscript. 2. Could the authors discuss whether a third vaccination might further increase the antibody titers in SARS-CoV-2-navie individuals?
Reviewer #2 (Comments for the Author): The authors determined abs titers only twice: 2 weeks and 2 months after first vaccination. Why the plasma samples were not tested at the same time after each of vaccination doses? The paper would benefit of a thorough English language correction as there are sentences that are difficult to follow. Some specific points: Line 23 -phrase "appropriate" -this term is not clear Line 36-38 -the sentence is not clear Line 52 -the authors write that "The time between infection and vaccination has been found to be 1-3 months" -what do they mean by that?
Reviewer #3 (Comments for the Author): The manuscript is written in standard English but there are a few lines that should be reviewed. Please check lines 25-26 (All participants were planned two doses of....); line 36 ("While" should be deleted); line 53 (should be "individuals were involved in institutional outbreaks). The author should include any reference/s regarding the approval documents (IRB protocol) by the ethics committee of each hospital if available.
The authors state the U/ml obtained in the study. It should be indicated the linear range of the detection method; for Elecsys anti-SARS-CoV-2 S, I think is 0.4 to 250 U/ml. The Discussion Section should be expanded. Please, could you discuss the current role of antibody detection and the potential role of neutralizing antibodies in protective immunity?
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In the manuscript, the authors analyzed the titers of Receptor binding domain of SARS-CoV-2 Spike protein-directed antibody in people with a history of COVID-19 or SARS-CoV-2-naive individuals after one and two doses of BNT162b2 vaccine. Based on the data obtained from a small size of samples, the author found that a two doses of BNT162b2 vaccine increased the antibody titers in SARS-CoV-2-naive individuals, while the second vaccination did not significantly increase the antibody titers in individuals with a history of COVID-19.The author's finding is supported by their clear data, although the data was obtained from a small size of samples as also discussed by the authors. The experiment and methodology of the manuscript are technically sound.
Specific comments are below. Therefore, we revised the Discussion section as follows: Thus, a single-dose vaccination might be sufficient in recently infected individuals. We also found that antibody titers began to wane at 6 months after the initial series of vaccinations (data not shown); however, this does not necessarily indicate a decrease in neutralizing activity (7). A booster dose of BNT16b2 could have immunogenicity (8), but the timing and recipient prioritization remain controversial (9).

Reviewer #2
The authors determined abs titers only twice: 2 weeks and 2 months after first vaccination. Why the plasma samples were not tested at the same time after each of vaccination doses?
Reply: Since IgG is generally produced 2 weeks after antigen exposure, the initial reaction was confirmed at 2 weeks after the first vaccination.
Concerning the timing of the second measurement, our purpose was to determine the final immunity established by the 2-dose vaccination, but not to compare the degree of first and second reactions. We therefore determined the antibody titers at 2 months after the first vaccination.
The paper would benefit of a thorough English language correction as there are sentences that are difficult to follow.
Reply: Thank you for your kind suggestion. A thorough English language review of the paper was completed by native speakers.
Some specific points: Line 23 -phrase "appropriate" -this term is not clear Line 52 -the authors write that "The time between infection and vaccination has been found to be 1-3 months" -what do they mean by that?
Reply: We understand that this is confusing and have made changes to reflect this: Two BNT162b2 vaccination doses were administered within 3 months of COVID-19 outbreak (between December 2020 and February 2021). .

Reviewer #3
The manuscript is written in standard English but there are a few lines that Therefore, we added the following sentence into the Discussion section: Thus, a single-dose vaccination might be sufficient in recently infected individuals. We also found that antibody titers began to wane at 6 months after the initial series of vaccination (data not shown); however, this does not necessarily mean a decrease in neutralizing activity (7). A booster dose of Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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