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. 2021 Sep 20;16(9):e0257668. doi: 10.1371/journal.pone.0257668

Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects?

Masaaki Takeuchi 1,*, Yukie Higa 1, Akina Esaki 1, Yosuke Nabeshima 2, Akemi Nakazono 1
Editor: Nicole J Moreland3
PMCID: PMC8452080  PMID: 34543337

Abstract

Background

Adverse reactions are more common after the second injection of messenger RNA vaccines such as Pfizer/BioNTech’s BNT162b2. We hypothesized that the degree and severity of reactogenicity after the second injection reflects the magnitude of antibody production against the SARS CoV-2 virus spike protein (spike IgG).

Methods and results

Blood samples were obtained from 67 Japanese healthcare workers three weeks after the first injection and two weeks after the second injection of the BNT162b2 vaccine to measure spike IgG levels. Using questionnaires, we calculated an adverse event (AE) score (0–11) for each participant. The geometric mean of spike IgG titers increased from 1,047 antibody units (AU/mL) (95% confidence interval (95% CI): 855–1282 AU/mL) after the first injection to 17,378 AU/mL (95% CI: 14,622–20,663 AU/mL) after the second injection. The median AE score increased from 2 to 5. Spike IgG levels after the second injection were negatively correlated with age and positively correlated with spike IgG after the first injection. AE scores after the second injection were not significantly associated with log-transformed spike IgG after the second injection, when adjusted for age, sex, AE score after the first injection, and log-transformed spike IgG after the first injection.

Conclusions

Although the sample size was relatively small, reactogenicity after the second injection may not accurately reflect antibody production.

Introduction

Effective and appropriate coronavirus disease 2019 (Covid-19) vaccination is the most promising strategy for controlling the spread of SARS-CoV2 infection on a worldwide basis [1–3]. Currently, nucleoside-modified messenger RNA (mRNA) vaccines encoding SARS-CoV2 full-length spike and adenoviral vector vaccines have been used mainly in Western countries. Vaccination levels in Canada, the United States, England, and Israel are now reaching 55%–69%, dramatically reducing the infection rate [4–7]. In Japan, the number of Covid-19 infections is increasing, but the vaccination rate is still low (26%) due to a limited supply of vaccines and a slow throughput for vaccine injection.

Local and systemic reactogenicity after the second injection of mRNA vaccines is more common than after the first injection [1, 2, 8]. The second injection has a booster effect that produces substantial antibody titers against SARS-CoV2 spike antigen. Thus, we hypothesized that the degree of reactogenicity following the second injection could be an indicator of the level of SARS-CoV2 spike antibody production. Accordingly, the aim of this study was to investigate whether local and systemic reactogenicity after the second injection of an mRNA vaccine reflects subsequent SARS-CoV2 spike antibody levels in Japanese healthcare workers.

Materials and methods

Study participants

This was a prospective, longitudinal, observational study in a single center. The study was approved by the institutional review board of the University of Occupational and Environmental Health, School of Medicine (approval number: UOEHCRB21-023). During early 2021, the only vaccine available in Japan was the BNT162b2 mRNA Covid-19 vaccine (Pfizer/BioNTech). The Japanese government started to distribute this vaccine to healthcare workers in February 2021, and our university hospital received the vaccine in the middle of March 2021. The hospital chairman decided to administer the first dose of BNT162b2 mRNA Covid-19 vaccine (30 μg per dose injected into the deltoid muscle) to hospital employees during the fourth week of March and the second dose during the third week of April (April 12 to April 16). Since we received ethical approval for the study on April 12 the time available to acquire informed consent and to conduct the study was quite short (range: 1 to 5 days), we advertised for the study participation to subjects working in four departments (Department of Laboratory and Transfusion Medicine, Department of Pathology, Department of Pharmacy, Second Department of Internal Medicine) in the hospital. Study participation was voluntary. Participant recruitment was started on April 12 and ended on April 16. Written informed consent was obtained from all personnel who agreed to participate.

Antibody test

Blood samples were obtained from participants before the second dose of the BNT162b2 mRNA vaccination (median: 20 days [interquartile range (IQR): 20 to 21 days] after the first dose) and 2 weeks after the second dose (median: 13 days [IQR: 11 to 14 days] after the second dose). A SARS-CoV2 IgG assay was performed with chemiluminescent immunoanalysis of microparticles used for quantitative detection of IgG antibodies against the spike protein of the SARS CoV-2 virus (spike IgG) employing the Architect system (Abbott Diagnostics) with a cut-off <50 antibody units/mL (AU/mL) in both blood samples. To exclude the possibility of previous Covid-19 infection, we also measured IgG antibodies against the nucleocapsid protein of Covid-19 (Abbott Diagnostics) using the second blood sample. The negative cut-off value for anti-nucleocapsid protein IgG was < 1.4 AU/mL.

Reactogenicity

All participants were asked to respond to questionnaires after both the first and second blood samples regarding local and systemic adverse effects to the vaccine injections. Questionnaires included a previous history of allergic reactions, severe adverse effects such as anaphylactic reactions, local reactogenicity such as pain at the injection site, swelling, lymph node swelling, and systemic reactogenicity, including fatigue, headache, myalgia, arthralgia, chills, and fever (body temperature ≥ 38°C). To estimate reactogenicity, we calculated an adverse effect score (AE score). For each symptom present, whether local and systemic, we assigned a score of 1. The exception was fever. A score of 1 was given if body temperature increased to ≥38.0°C but <38.4°C. A score of 2 corresponded to a body temperature of ≥38.4°C to <38.9°C. Development of a body temperature ≥38.9°C was scored as 3 [2, 3]. We summed all scores and calculated AE scores after the first and second injection (range: 0 to 11).

The primary outcome was to determine whether the AE score after the second injection was associated with spike IgG levels two weeks after the second injection.

Statistical analysis

Levels of spike IgG were expressed as the median, IQR, and the geometric mean and 95% confidence intervals calculated using Student’s t test distribution on log-transformed data. AE score was shown as the median and IQR. Categorical data are presented by number and percentage. Given the small number of participants, we did not perform formal statistical comparisons between groups. Spearman’s correlation analysis was used to compare spike IgG levels after the first and second injections and between spike IgG after the second injection and age. Correlation analysis was further performed on subgroups stratified by gender. To predict the level of log-transformed spike IgG two weeks after the second injection, multivariate linear regression analysis was performed, using age, sex, log-transformed spike IgG three weeks after the first injection, and AE scores after the first and the second injection as covariates. A two-sided p-value < 0.05 was considered significant. All statistical analyses were conducted using R software version 4.0.4 (R foundation for Statistical Computing, Vienna).

Results

Among 109 healthcare workers who were working in any of the four Departments, 69 subjects agreed to participate the study. However, two participants did not come to receive a second blood sample examination within the specified time window; thus, we excluded them from the analysis. The final study population comprised 67 participants (age 43 years [IQR: 32–51 years], 21 males). Six subjects reported previous histories of allergic reactions.

Reactogenicity

No one developed a severe adverse reaction after the vaccinations. Fig 1 presents local and systemic reactogenicity after the first and second injections. Except for myalgia, the prevalence of all other reactogenicities increased from the first injection to the second injection, resulting in a higher AE score after the second injection (median: 5, IQR: 2–6) than after the first injection (median: 2, IQR: 1–3). Since previous studies showed higher reactogenicities in younger subjects [2, 9], we divided subjects into two groups stratified by age and compared reactogenicities after the second injection. Reactogenicity was higher among subjects < 50 years than among those ≥ 50 years (Table 1). Thus, AE scores after the second injection were higher in subjects who were < 50 years (median: 5, IQR: 4–6) compared with subjects ≥ 50 years (median: 2, IQR: 1–3).

Fig 1. Local and systemic reactions after the first and second injections of the Pfizer/BioNTech BNT162b2 vaccine among all subjects.

Fig 1

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Fever was defined as a body temperature ≥ 38°C after a vaccine injection.

Table 1. Comparisons of adverse reaction and spike IgG titers after the second injection of BNT162b2 mRNA vaccine among subjects ≤ 50 years and those > 50 years.

Variable Age ≤ 50 years (n = 46) Age > 50 years (n = 21)
Male gender 14 (30%) 7 (33%)
Spike IgG (AU/mL) 20,070 (IQR: 13529–33898) 14,766 (10131–19693)
Pain at injection site 42 (91%) 17 (81%)
Swelling 23 (50%) 5 (24%)
LN swelling 10 (22%) 2 (9.5%)
Fatigue 37 (80%) 7 (33%)
Headache 28 (61%) 5 (24%)
Myalgia 32 (70%) 12 (57%)
Chills 26 (57%) 3 (14%)
Arthralgia 18 (39%) 2 (9.5%)
fever 13 (28%) 2 (9.5%)
Degree of fever
< 38.0°C 33 (72%) 19 (90%)
≥ 38.0 but < 38.4°C 7 (15%) 2 (10%)
≥ 38.4 but < 38.9°C 3 (6.5%) 0 (0%)
≥ 38.9°C 3 (6.5%) 0 (0%)
AE score 5.0 (4.0–6.0) 2.0 (1.0–3.0)
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Continuous values are expressed as median and interquartile range (IQR).

AE, adverse effect.

Antibodies

None of the subjects were positive for anti-nucleocapsid IgG (median: 0.04 AU/mL, IQR: 0.02–0.07 AU/mL). All subjects were positive for spike IgG (> 50 AU/mL), for which the median, IQR, geometric mean and its 95% confidence interval after the first injection were 1,065 AU/mL, 741–1,693 AU/mL, 1,047 AU/mL, and 855–1,282 AU/mL, respectively. After the second injection, corresponding values increased to 17,259 AU/mL, 12,088–28,999 AU/mL, 17,378 AU/mL, and 14,622–20,663 AU/mL, respectively, a 16-fold increase of spike IgG from the first to the second injection. There was a positive correlation between spike IgG levels after the first injection and after the second injection (r = 0.62, p < 0.001, Fig 2A). There was also a negative correlation between age and spike IgG levels after the second injection (r = -0.37, p = 0.002, Fig 2B).

Fig 2.

Fig 2

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A. A linear correlation between spike IgG level after the first injection and that after the second injection in all subjects (left), in female subjects (middle), and in male subjects (right). B. A linear correlation between age and spike IgG level after the second injection in all subjects (left), in female subjects (middle), and in male subjects (right). sIgG, spike IgG.

Linear regression analysis

Univariate linear regression analysis revealed that age, log-transformed spike IgG after the first injection, and AE score after the second injection were associated with log-transformed spike IgG after the second injection. However, sex and AE score after the first injection was not associated with log-transformed spike IgG after the second injection (Table 2).

Table 2. Univariate linear regression analysis for the association of log-transformed spike IgG two weeks after the second injection of BNT162b2 mRNA vaccine.

variables Beta coefficients (95% CI) SE t-value p-value
age -0.010 (-0.016 –-0.004) 0.003 -3.167 0.002
male gender -0.130 (-0.290–0.030) 0.080 -1.622 0.110
log spike IgG after the first injection 0.636 (0.497–0.775) 0.070 9.133 <0.001
AE score after the first injection 0.025 (-0.026–0.076) 0.026 0.967 0.337
AE score after the second injection 0.057 (0.030–0.085) 0.014 4.119 <0.001
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AE, adverse effect; CI, confidence interval; SE, standard error.

Multivariate linear regression analysis revealed that age and log-transformed spike IgG after the first injection were significantly associated with log-transformed spike IgG after the second injection. However, sex, AE score after the first injection, and AE score after the second injection were not significantly associated with log-transformed spike IgG after the second injection (Table 3). After removing those three variables from the model, the final model yielded an F-statistic of 52.4, a residual standard error of 0.29, and an adjusted R2 of 0.61.

Table 3. Multivariate linear regression analysis of the association of log-transformed spike IgG two weeks after the second injection of BNT162b2 mRNA vaccine.

variables Beta coefficients (95% CI) SE t-value p-value
age -0.006 (-0.010 –-0.001) 0.002 -2.640 0.011
male gender -0.093 (-0.199–0.012) 0.053 -1.772 0.081
log spike IgG after the first injection 0.550 (0.412–0.688) 0.069 7.977 <0.001
AE score after the first injection 0.001 (-0.035–0.036) 0.018 0.031 0.976
AE score after the second injection 0.017 (-0.006–0.041) 0.012 1.491 0.141
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Residual standard error: 0.1879.

Adjusted R2: 0.6285.

F-statistic: 23.33, p<0.001.

Discussion

The main findings of this study can be summarized as follows: (1) the prevalence of local and systemic reactogenicity increased approximately 2.5 times after the second injection, compared to the first injection, and its severity was more significant in younger people; (2) spike IgG titers became positive in all participants after the first injection, and those titers increased 16-fold after the second injection; (3) spike IgG titers after the second injection were negatively correlated with age, and positively correlated with spike IgG after the first injection; (4) multivariate linear regression analysis revealed that the degree of reactogenicity after the second injection was not significantly associated with spike IgG levels after the second injection after adjusting for age, gender, AE score after the first injection, and spike IgG levels after the first injection.

None of the subjects in this study showed positive results for nucleocapsid IgG; thus, we believe that all participants were Covid-19 naïve subjects [10]. Although the degree of adverse effects after the first injection was similar to those in a previous publication from western countries [2, 3, 11], the prevalence of reactogenicity after the second injection was higher. Although proposed causes may be related to racial differences and geographic differences of non-COVID-19 corona virus exposure, we should consider a fixed dosing regimen as a potential cause of different prevalence of adverse reactions. Dose-dependent increases in adverse effects have been observed with mRNA vaccines [1–3] and the current recommended dose of BNT162b2 mRNA vaccine (30 μg), which was determined for western subjects, would result in a higher dose for people with smaller body mass, such as Japanese.

The geometric mean of spike IgG after the second injection was 17,378 AU/mL, which was higher than corresponding values measured in a naïve Spanish COVID-19 population [12], but similar to another study [13]. In addition, the range of values was quite broad, from 2,826 AU/mL to 70,272 AU/mL. Although there is no definite cut-off of value for spike IgG sufficient to prevent COVID-19 infection, the FDA authorized only the use of high-titer COVID-19 convalescent plasma (spike IgG > 840 AU/mL), for treatment of patients hospitalized with COVID-19 infection, early in the course of the pandemic [14]. Prendecki et al. reported that one COVID-19 naïve individual whose spike IgG titer was 61.8 AU/mL, developed symptomatic COVID-19 infection 5 weeks after the first dose of BNT162b2 vaccine [15]. The authors also stated that spike IgG titers < 250 AU/mL might not be sufficient for virus neutralization. The lowest IgG titers observed in this study exceeded the latter value by nearly 10-fold. Spike IgG levels after the second injection were higher in subjects < 50 years than in those ≥ 50 years. Previous studies did not show age-dependent efficacy of BNT162b2 for subsequent COVID-19 infection [3, 6]. Thus, these results raise concerns regarding the recommended dose of BNT162b2 mRNA vaccine in younger Japanese people. The current infection situation in Japan makes dose response trials with BNT162b2 vaccine almost impossible. However, if the level of spike IgG three weeks after the first vaccination is > 1,000 AU/mL, the second injection dose could be reduced in younger people, which would enhance the Japanese vaccination rate.

Although efficacy and adverse effects of vaccines appear opposite, they may be related via the strength of the immune response to the vaccine. However, there was no significant association between AE score after the second injection and spike IgG levels after adjusting for age, gender, AE score after the first injection, and spike IgG level after the first injection. Several recent publications and pre-print articles have addressed the relationship between adverse effects and antibody productions. Müller et al. [9] demonstrated that spike IgG and neutralizing antibody titers were significantly lower in patients with > 80 years old than in patients with < 60 years old. Although the prevalence of adverse effects after vaccination was lower in older persons (>80 years old) compared with younger recipients (< 60 years old), there was no correlation between spike IgG levels and post-vaccination adverse effects. Coggins and colleagues [16] calculated AE score and measured spike IgG titers in 206 healthcare workers. AE score was correlated with age, sex, and body weight. However, spike IgG titers were not correlated with AE score after the first injection or the second injection. Debes et al. [17] longitudinally collected spike IgG antibody levels and symptoms after vaccination with either the Moderna or Pfizer/BioNTech vaccines in 954 healthcare workers, and found that clinically significant symptoms (fever, chills, fatigue) were independently associated with higher median IgG measurements after adjusting for elapsed time after the second vaccination. However, the ratio of IgG antibody measurements was not remarkably different between the two groups (8.82 in subjects with significant symptoms vs. 8.46 in those with no or mild symptom). The authors concluded that regardless of vaccine reactions, either spike mRNA vaccine provides a robust spike antibody response. These studies concur that adverse effects after vaccination do not necessarily reflect subsequent spike IgG production, and more importantly, lack of an adverse effect after the vaccination does not mean that the vaccine did not work effectively [16].

Although the relatively small sample size could produce false negative results, adverse effects after the second injection may be dose-dependent immune reactions after repeated exposure to excipients to stabilize lipid nanoparticles, such as PEG2000, which serves as the vehicle for the vaccine mRNA [8]. Age was negatively correlated with spike IgG production, which agrees with previous studies [3, 9]. Spike IgG levels after the first injection were also positively correlated with spike IgG production after the second injection. However, the model including age and spike IgG after the first vaccination was not sufficiently robust to predict the amount of spike IgG after the second injection with confidence.

Study limitations

Small sample size limited the generalizability of our results. Since our results were derived from healthy Japanese healthcare workers, further studies should examine subjects with comorbidities and more aged populations [18, 19]. A longitudinal study to determine spike IgG levels after vaccination is another important topic for ongoing research.

Conclusions

Reactogenicity after the second injection of BNT162b2 mRNA vaccine was common, especially in young to middle aged Japanese healthcare workers. However, its intensity was not closely correlated with subsequent spike IgG levels, after adjusting for age, sex, reactogenicity after the first injection, and spike IgG levels after the first injection. Thus, reactogenicity after the second injection is probably not a reliable measure of antibody production.

Supporting information

S1 File. Data file.

(XLSX)

Click here for additional data file. (12.6KB, xlsx)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

The authors received no specific funding for this work.

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Nicole J Moreland

12 Aug 2021

PONE-D-21-19170

Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects?

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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: I Don't Know

**********

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

**********

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: Review of “Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects, Manuscript PONE-D_21-19170"

Page 3, Line 46 “produces a substantial antibody titers” should be either produces substantial antibody titres or produces a substantial antibody titer.

Page 5, Lines 89-94 It took me a longish time to understand the scoring system and then not entirely sure I’ve got it right. I’m assuming for each symptom present there was a score of one; with different measured levels of fever being the exception. I wonder if it’s worth adding a couple of examples of symptom combinations and the resulting score? Alternatively suggest the following wording

Instead of “When local and systemic reactogenicity, except for fever, was present, we scored it as 1.”

Maybe- For each symptom present, whether local or systemic, we assigned a score of 1. The exception was fever...

Reviewer #2: The authors have conducted a small but nonetheless interesting trial of adverse reactions following Pfizer/BioNTech’s BNT162b2 vaccination and asked whether an increase in adverse reactions could relate to an increased reactogenictiy and thus higher antibody response.

Most of the findings of the paper are in agreement with previous studies and the results are interesting. Although as the authors point out, the low numbers included in the trial would need to be validated in larger trials. I note that there are a couple of pre-prints showing the same findings in larger trials such as:

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers, in MedRxiv.

This could be cited in the papers.

The one comment I have is to the statement:

Given the small number of participants, we did not perform formal statistical comparisons between groups.

I would suggest the authors do at least present statistical comparisons and report p-values for correlations, as I suspect the negative correlation between age and IgG levels after second vaccine would not be statically significant, which would need to be discussed further. This observation is backed up by other studies but may not be significant here due to smaller sample size.

**********

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Reviewer #1: Yes: Dr Janine Paynter

Reviewer #2: No

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PLoS One. 2021 Sep 20;16(9):e0257668. doi: 10.1371/journal.pone.0257668.r002

Author response to Decision Letter 0

22 Aug 2021

Dear Editors and Reviewers,

We thank all of you for your thoughtful and constructive comments regarding our manuscript entitled,” Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects? (PONE-D-21-19170)”. Below, in blue, we have provided a point-by-point response to your comments. Red denotes new or revised text in the manuscript

Responses to the Editors:

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.

RESPONSE: We checked that references are complete and correct. Since four pre-prints (medRxiv) cited in the previous manuscript have now been published (Müller, Mazzoni, Velasco, and Narasimham), we replaced the following references:

9. Müller L, Andrée M, Moskorz W, Drexler I, Walotka L, Grothmann R, et al. Age-dependent Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination. Clin Infect Dis. 2021:ciab381. doi: 10.1093/cid/ciab381. PubMed PMID: 33906236; PubMed Central PMCID: PMC8135422.

10. Mazzoni A, Di Lauria N, Maggi L, Salvati L, Vanni A, Capone M, et al. First dose mRNA vaccination is sufficient to reactivate immunological memory to SARS-CoV-2 in subjects who have recovered from COVID-19. J Clin Invest. 2021;131(12):e149150. doi: 10.1172/JCI149150. PubMed PMID: 33939647; PubMed Central PMCID: PMC8203460.

12. Velasco M, Galán MI, Casas ML, Pérez-Fernández E, Martínez-Ponce D, González-Piñeiro B, et al. Impact of previous coronavirus disease 2019 on immune response after a single dose of BNT162b2 severe acute respiratory syndrom coronavirus 2 vaccine. Open Forum Infect Dis. 2021;8(7). doi: 10.1093/ofid/ofab299. PubMed PMID: 34258322; PubMed Central PMCID: PMC8244747.

13. Narasimhan M, Mahimainathan L, Araj E, Clark AE, Markantonis J, Green A, et al. Clinical Evaluation of the Abbott Alinity SARS-CoV-2 Spike-Specific Quantitative IgG and IgM Assays among Infected, Recovered, and Vaccinated Groups. J Clin Microbiol. 2021;59(7):e0038821. doi: 10.1128/JCM.00388-21. PubMed PMID: 33827901; PubMed Central PMCID: PMC8218760.

We added two references in response to the comments from reviewer #2.

16. Coggins SAA, Laing ED, Olsen CH, Goguet E, Moser M, Jackson-Thompson BM, et al. Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers. medRxiv [Preprint]. 2021. doi: https://doi.org/10.1101/2021.06.25.21259544.

17. Debes AK, Xiao S, Colantuoni E, Egbert ER, Caturegli P, Gadala A, et al. Association of Vaccine Type and Prior SARS-CoV-2 Infection With Symptoms and Antibody Measurements Following Vaccination Among Health Care Workers. JAMA Intern Med. 2021. doi: 10.1001/jamainternmed.2021.4580. PubMed PMID: 34398173.

Response to the reviewer #1:

Page 3, Line 46 “produces a substantial antibody titers” should be either produces substantial antibody titers or produces a substantial antibody titer.

RESPONSE: Thank you for pointing this out. We changed this to “produces substantial antibody titers”.

Page 3, Lines 46-47:

The second injection has a booster effect that produces substantial antibody titers against SARS-CoV2 spike antigen.

Page 5, Line 89-94 It took me a longish time to understand the scoring system and then not entirely sure I’ve got it right. I’m assuming for each symptom present there was a score of one; with different measured levels of fever being the exception. I wonder if it’s worth adding a couple of examples of symptom combinations and the resulting score? Alternatively suggested the following wording

Instead of “When local and systemic reactogenicity, except for fever, was present, we score it as 1.”

Maybe- For each symptom present, whether local and systemic, we assigned a score of 1. The exception was fever…

RESPONSE: Thank you for the thoughtful comments. We agree that the weight of each symptom may be different. However, we also don’t know which symptom is more important. Thus, we arbitrarily scored for adverse effects. A similar scoring system has been used in the recent publications (ref. #16), but definitions vary among studies (ref. #16, #17). We corrected the sentences as suggested.

Page 5, Lines 93-94

For each symptom present, whether local and systemic, we assigned a score of 1. The exception was fever. A score  

Responses to the Reviewer #2:

The authors have conducted a small but nonetheless interesting trial of adverse reactions following Pfizer/BioNTech’s BNT162b2 vaccination and asked whether an increase in adverse reactions could relate to an increased reactogenicity and thus higher antibody response.

Most of the findings of the paper are in agreement with previous studies and the results are interesting. Although as the authors pointed out, the low numbers included in the trial would need to be validated in larger trials. I note that there are a couple of pre-prints showing the same findings in larger trials such as:

Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers, in MedRxiv. This could be cited in the papers.

RESPONSE: Thank you for the positive comments and important suggestions. We incorporated recent publications or pre-prints in this revised manuscript and discussed reactogenicity and antibody production.

Page 13-14, Discussion, Lines 239-259:

Several recent publications and pre-print articles have addressed the relationship between adverse effects and antibody productions. Müller et al. [9] demonstrated that spike IgG and neutralizing antibody titers were significantly lower in patients with > 80 years old than in patients with < 60 years old. Although the prevalence of adverse effects after vaccination was lower in older persons (>80 years old) compared with younger recipients (< 60 years old), there was no correlation between spike IgG levels and post-vaccination adverse effects. Coggins and colleagues [16] calculated AE score and measured spike IgG titers in 206 healthcare workers. AE score was correlated with age, sex, and body weight. However, spike IgG titers were not correlated with AE score after the first injection or the second injection. Debes et al. [17] longitudinally collected spike IgG antibody levels and symptoms after vaccination with either the Moderna or Pfizer/BioNTech vaccines in 954 healthcare workers, and found that clinically significant symptoms (fever, chills, fatigue) were independently associated with higher median IgG measurements after adjusting for elapsed time after the second vaccination. However, the ratio of IgG antibody measurements was not remarkably different between the two groups (8.82 in subjects with significant symptoms vs. 8.46 in those with no or mild symptom). The authors concluded that regardless of vaccine reactions, either spike mRNA vaccine provides a robust spike antibody response. These studies concur that adverse effects after vaccination do not necessarily reflect subsequent spike IgG production, and more importantly, lack of an adverse effect after the vaccination does not mean that the vaccine did not work effectively [16].

Page 17, References, Lines 344-351:

16. Coggins SAA, Laing ED, Olsen CH, Goguet E, Moser M, Jackson-Thompson BM, et al. Adverse effects and antibody titers in response to the BNT162b2 mRNA COVID-19 vaccine in a prospective study of healthcare workers. medRxiv [Preprint]. 2021. doi: https://doi.org/10.1101/2021.06.25.21259544.

17. Debes AK, Xiao S, Colantuoni E, Egbert ER, Caturegli P, Gadala A, et al. Association of Vaccine Type and Prior SARS-CoV-2 Infection With Symptoms and Antibody Measurements Following Vaccination Among Health Care Workers. JAMA Intern Med. 2021. doi: 10.1001/jamainternmed.2021.4580. PubMed PMID: 34398173.

The one comment I have is to the statement:

Given the small number of participants, we did not perform formal statistical comparisons between groups.

I would suggest the authors do at least present statistical comparisons and report p-values for correlations, as I suspect the negative correlation between age and IgG levels after second vaccine would not be statistically significant, which would need to be discussed further. This observation is backed up by the other studies but may not be significant here due to smaller sample size.

RESPONSE: Thank you for the thoughtful comments. The reason why we did not perform statistical comparisons between groups (Table 1) is a problem of “multiplicity”. Our primary endpoint was to determine whether the severity of adverse effects after the vaccination predicts the amount of antibody production. Thus, we only performed statistical analysis for this purpose. However, we agreed with your comments and incorporated p-values for correlations in this revised manuscript. Although the number of subjects was small, we found that the correlation between age and spike IgG after the second vaccination was statistically significant (p=0.002). We incorporated the results in the manuscript text, and in Figure 2.

Page 9, Lines 154-157:

There was a positive correlation between spike IgG levels after the first injection and after the second injection (r = 0.62, p < 0.001, Fig 2A). There was also a negative correlation between age and spike IgG levels after the second injection (r = -0.37, p = 0.002, Fig 2B).

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Decision Letter 1

Nicole J Moreland

8 Sep 2021

Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects?

PONE-D-21-19170R1

Dear Dr. Takeuchi,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nicole J. Moreland

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All comments have been addressed in the revised version and I am happy to accept this paper for publication.

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 #2: 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 #2: Yes

**********

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

Reviewer #2: 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 #2: 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 #2: 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 #2: (No Response)

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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 #2: No

Acceptance letter

Nicole J Moreland

10 Sep 2021

PONE-D-21-19170R1

Does reactogenicity after a second injection of the BNT162b2 vaccine predict spike IgG antibody levels in healthy Japanese subjects?

Dear Dr. Takeuchi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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