Anti‐spike protein IgG antibody responses up to 3 months after the third dose of the BNT162b2 mRNA vaccine in medical care workers

Abstract Background This study aimed to assess the changes in anti‐spike protein IgG antibody titer over time following mRNA vaccination (BNT162b2) against severe acute respiratory syndrome coronavirus 2. Methods We monitored IgG levels in 23 medical care workers (MCWs) for up to 3 months after administering the third dose of BNT162b2. Blood samples were periodically collected from all participants. Results Following the third dose, the median antibody titer increased to 252 and 327% compared with antibody levels at 1 and 3 months after the second dose, respectively. Additionally, compared with 1 month after the second dose, the median antibody titer decreased to 30.2 and 9.8% at 3 and 6 months, respectively, and to 39.1% at 3 months, compared with 1 month after the third dose. Conclusion Antibody levels declined quickly after the second dose but declined more slowly after the third dose, showing a booster effect. This study provides insights into the immunogenicity of booster doses and time intervals for booster vaccination strategies.

| 141 HIRANO vaccines against the Omicron variant.The seventh vaccination began in September 2023 as a monovalent vaccine.
The mRNA vaccination program is a novel initiative in Japan, sparking diverse opinions regarding its efficacy and safety among ordinary people as well as doctors.This study was undertaken because of the absence of any published reports on immunoglobulin G (IgG) antibodies after mRNA vaccination in Japan at that time.In addition, this study was initiated concurrently with vaccination work to emphasize Evidence-Based Medicine among our staff in the clinical setting.Considering that some of the staff in my clinic had reservations with regard to vaccination, I was concerned that the vaccination process would not proceed smoothly.
The plaque reduction neutralization test (PRNT), a virus neutralization test, is the gold standard for evaluating vaccine efficacy.
However, because PRNT requires expertise and time, commercial serological assays are commonly used, which are simpler, low-cost, and more practical.Among the SARS-CoV-2 structural proteins, the spike and nucleocapsid proteins are the major immunogens.Since the anti-spike protein (S1 subunit RBD) IgG antibodies correlate with neutralizing antibodies, 5,6 they are measured to evaluate humoral immune responses after vaccination.
In studies of humoral immunity based on IgG antibody levels, the timing of antibody level measurement after vaccination is crucial.
Numerous studies have documented varying decay dynamics of IgG antibodies following COVID-19 booster vaccinations. 7,8However, the precise nature of this decay remains unclear.Some crucial aspects of antibody research include assessing the immunogenicity of vaccines, determining protective thresholds, 9 and studying adverse reactions. 10spite these goals, antibody titers diminish naturally over time, with an especially rapid initial decay rate that can introduce interval bias to the measurements.To mitigate such bias, this study employed a more precise timing for blood sample collection than prior research endeavors.

| Study participants, ethics statement, and vaccine administration
The BNT162b2 vaccination was planned for MCWs from the Fukui Health Cooperative Association (FHCA) from April to June 2021.This study was reviewed and approved by the FHCA Ethics Committee in February 2021 (approval number F21001).All procedures were in accordance with the Declaration of Helsinki.In this study, 469 MCWs from FHCA were called to participate, 23 of whom applied.
The protection of personal information and voluntary participation in the study was explained, and written informed consent was obtained from all study participants.
All MCWs received the BNT162b2 vaccine.The first vaccine was administered between April 4 and June 4, 2021, the second dose after 3 weeks, and the third dose8 months after the first (median: 237 d, interquartile range [IQR]: 234-270 d).

| Antibody measurement
The ARCHITECT SARS-CoV-2 IgG II Quant assay (Abbott) was used for IgG antibody level measurements.This assay is a chemiluminescent microparticle immunoassay (CMIA) used for the qualitative and quantitative determination of IgG antibodies against SARS-CoV-2 in human serum and plasma on the ARCHITECT i System.
The assay has also been used to evaluate humoral immunity in infected individuals and immunogenicity in individuals who have received mRNA vaccines. 11The positive cut-off antibody level was defined as 50.0 AU/mL as per the manufacturer's instructions.The test sensitivity and specificity were defined at 98.1 and 99.6%, respectively, as per the U.S. Food and Drug Administration. 12Blood samples were periodically collected from the participants, and the antibody measurements were outsourced to a commercial laboratory (BML Inc.; Figure 1).

| Statistics analysis
The results are presented as the median and interquartile range Table 1 shows the number of participating MCWs, the period after the first, second, and third doses, and the antibody levels.Only small data scattering was observed between vaccination and antibody testing.
Compared with 1 month after the second dose, the median antibody titer at 3 and 6 months decreased to 30.2% (p < 0.0001) and 9.8% (p < 0.0001), respectively.In addition, there was no significant difference in the antibody titer 6 months after the second dose compared with the antibody titer 1 week before administration of that dose (p = 0.11).
The median antibody titer after the third dose was 18,000 AU/ mL (IQR:12000-25,000) after 1 month and 7040 AU/mL (IQR:4152-10,250) after 3 months.In addition, compared with 1 month after the third dose, the median antibody titer at 3 months decreased to 39.1% (p < 0.01).Compared with one and 3 months after the second dose, the median antibody titer increased to 252% (p < 0.01) and 327% (p < 0.001) at 1 and 3 months after the third dose, respectively (Figure 2).

| DISCUSS ION
Vaccines represent the most effective weapon to control the COVID-19 pandemic, and monitoring their effectiveness is critical for assessing individual protection against SARS-CoV-2 infection.
Vaccine protection against SARS-CoV-2 infection wanes over time, necessitating the administration of updated booster doses.The effectiveness of booster vaccination depends on the optimal vaccine dose, timing of vaccination, and the pattern of antibody response decay.Additionally, as the type of vaccine, sample collection interval, and type of assay vary from study to study, actual data cannot be easily compared.Table 2 shows several studies that have used methods similar to those in this study.The nine prospective studies listed all targeted MCWs and had relatively small sample sizes with a median of 30 (IQR: 10.5-108).Research with a large sample size was difficult to conduct because the novel coronavirus continued to spread in 2021, and the medical field was under pressure, making it difficult set aside sufficient research capacity.
In the present study, compared with 1 month after the second dose, the median antibody titer decreased to 30 and 9.8% at 3 and 6 months, respectively.Other studies indicated a decrease in IgG antibody titer to 26% and 30.8% at 3 months compared to 1 month after the second dose, 8,13 comparable to the results of the present study.In addition, antibody titer decreased to 7.2%, 7.5%, 10.4%, and 11.1% at 6 months compared with those at 1 month after the second dose (Table 2). 7,8,14,15In contrast, 3 months after the second dose, the median antibody titer decreased to 30% compared with 1 month later.Three months after the third dose, the median antibody titer decreased to 39% compared with 1 month later.In addition, the antibody titer 1 and 3 months after the third dose increased 2.5-fold and 3.3-fold, compared with the second dose, respectively.Therefore, the rate of decline in the antibody titer 3 months after the third dose was lower than that after the second dose.The increase in antibody titer after the booster shot and the decrease in decay rate indicates a booster effect.| 143 HIRANO Although our study was conducted up to 3 months after the third dose, a study of 3972 MCWs found that antibody levels declined more slowly 5 months after the third dose of the BNT162b2 vaccine than after the second dose, and the humoral response persisted for 5 months. 16A recent Japanese study has also shown that IgG antibody titers are more persistent after the third dose than after the second dose. 17Among the 1,137,804 adults aged ≥60 years who received their third dose in Israel, there was a significant reduction in confirmed SARS-CoV-2 infections and severe cases, demonstrating the effectiveness of the booster shot. 18moral immunity is enhanced by the reactivation of memory B cells by the third antigen stimulation with a vaccine or natural infection. 19Although not investigated in our study, few prospective cohort studies have investigated short-term responses related to peaks in antibody titer after BNT162b2 vaccination using the Abbott assay. 20,21These studies indicate that IgG antibody titers reach peak levels after 1-2 weeks after the second dose of mRNA vaccines.However, the short-term response associated with the peak antibody titer after each booster shot of the mRNA vaccine is poorly understood.The dynamics of IgG antibodies after mRNA vaccination have not been fully elucidated, and no mathematical model has been established to date.

IQR of antibody levels (AU/mL)
Quantitative antibody testing is essential for understanding humoral immunity in vaccinated individuals and populations; however, many testing methods and products exist.To enable the monitoring of antibodies, compatibility evaluation of 125 testing methods has also been performed by the World Health Organization. 22Methods for quantifying SARS-CoV-2 antibodies using automatic analysis devices include enzyme-linked immunosorbent assay, chemiluminescent and electrochemiluminescence immunoassays, and CMIA.
CMIA (Abbott assay) with high sensitivity and specificity was used in this study.This assay is also available for research in nonlaboratory, general clinical settings.
IgG antibody titers following mRNA vaccination are influenced by age, [23][24][25] gender, 24,26 BMI, 27 smoking, 26 history of diabetes, 28 and hypertension. 28Although this study targeted healthy MCWs, seven had hypertension and five were over 65 years of age.These demographic and clinical characteristics should be considered when evaluating antibody titers.
Serious adverse events were uncommon, and there were no safety concerns regarding the third dose of the BNT162b2 vaccine. 29nitoring anti-S-RBD IgG levels as a correlate of protection will help answer important questions regarding viral neutralization and immunity against SARS-CoV-2 in clinical settings.Understanding the dynamics of anti-S-RBD IgG will allow healthcare providers to optimize vaccine programs.
The strength of this prospective study was that blood samples were collected regularly from all participants.The limitation of this study is attributed to the small number of participants, 23 of 469 subjects.This is because of the nature of our facility (FHCA), which comprises several small workplaces focused on medical and nursing care.The lack of prior experience with prospective studies, along with limited publicity, contributed to this limitation.Although the significance of vaccines from the perspective of both efficacy and safety should be considered, this study did not examine safety-related issues, such as side effects.

| CON CLUS ION
Our prospective cohort study of 23 MCWs showed that compared with 1 month after the second dose, the median antibody titer decreased to 30.2% and 9.8% at 3 and 6 months, respectively.After

(
IQR) for continuous variables.The median values of the three groups, representing IgG antibody titers after the second dose, were compared using a nonparametric Wilcoxon signed-rank test.The two groups representing IgG antibody titers after the third dose and groups after the second and third doses were compared using the Wilcoxon rank sum test.The statistical significance level was set at p ≤ 0.05.F I G U R E 1 Timeline of vaccination and antibody measurement in MCWs.The number of days between vaccination and antibody measurement is presented as the median.In this prospective cohort study, 23 MCWs (8 males, 15 females) were tested for IgG antibodies.The 23 MCWs comprised three physicians, eight nurses, three pharmacists, four care workers, and five medical clerks, with a median age of 55 (IQR: 43-62 years).Of the 23 MCWs, seven had hypertension and one had diabetes, but none were smokers.The median body mass index was 22.8 (IQR: 20.8-25.8).The first test was performed before vaccination (median: 6 d, IQR: 4-9 d), and all participants were confirmed to be negative for antibodies (<50 AU/mL).Table 1 lists the subsequent testing periods.All 23 MCWs participated for up to 6 months after the second dose.However, five participants dropped out of the study 1 month after the third dose, and another four dropped out 3 months after the third dose.During the study period, 13 MCWs (57%) underwent polymerase chain reaction (PCR) testing after exposure to a patient with coronavirus disease 2019 (COVID-19).None of the MCWs had a positive PCR test nor exhibited an increased antibody titer.This result indicates that the MCWs were not infected with COVID-19.However, anti-nucleocapsid antibodies were not measured.

F I G U R E 2
Abbott (AU)