Article

Attenuation of Antibody Titers from 3 to 6 Months after theSecond Dose of the BNT162b2 Vaccine Depends on Sex, with
Age and Smoking Risk Factors for Lower Antibody Titers at
6 Months
Yushi Nomura 1,2 , Michiru Sawahata 1,3, * , Yosikazu Nakamura 4 , Ryousuke Koike 1,2 , Otohiro Katsube 1 ,Koichi Hagiwara 3 , Seiji Niho 2 , Norihiro Masuda 5 , Takaaki Tanaka 6 and Kumiya Sugiyama 1,7
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Citation: Nomura, Y.; Sawahata, M.;
Nakamura, Y.; Koike, R.; Katsube, O.;

7

Hagiwara, K.; Niho, S.; Masuda, N.;
Tanaka, T.; Sugiyama, K. Attenuation*

Department of Respiratory Medicine and Clinical Immunology, National Hospital Organization Utsunomiya
National Hospital, Utsunomiya 329-1193, Japan; uc.nomura@gmail.com (Y.N.);ryou.k.1207@gmail.com (R.K.); 5okatsube@gmail.com (O.K.); sugiyama@dokkyomed.ac.jp (K.S.)
Department of Pulmonary Medicine and Clinical Immunology, Dokkyo Medical University,
Shimotsuga 321-0293, Japan; siniho@dokkyomed.ac.jp
Department of Medicine, Division of Pulmonary Medicine, Jichi Medical University,
Shimotsuke 329-0498, Japan; hagiwark@me.com
Department of Public Health, Jichi Medical University, Shimotsuke 329-0498, Japan; nakamuyk@jichi.ac.jp
Department of Surgery, National Hospital Organization Utsunomiya National Hospital,
Utsunomiya 329-1193, Japan; norim0507@gmail.com
Department of Orthopaedic Surgery, National Hospital Organization Utsunomiya National Hospital,
Utsunomiya 329-1193, Japan; tanaka.takaaki.vh@mail.hosp.go.jp
Saitama Medical Center, Department of Respiratory Medicine and Clinical Immunology,
Dokkyo Medical University, Shimotsuga 321-0293, Japan
Correspondence: sawahata@jichi.ac.jp; Tel.: +81-285587350

of Antibody Titers from 3 to 6 Months
after the Second Dose of the
BNT162b2 Vaccine Depends on Sex,
with Age and Smoking Risk Factors
for Lower Antibody Titers at
6 Months. Vaccines 2021, 9, 1500.
https://doi.org/10.3390/vaccines
9121500
Academic Editor: Ger Rijkers
Received: 15 November 2021
Accepted: 15 December 2021
Published: 18 December 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Abstract: Objective: We aimed to determine antibody titers at six months and their percentage change
from three to six months after the second dose of the BNT162b2 coronavirus disease 2019 (COVID-19)
mRNA vaccine (Pfizer/BioNTech) and to explore clinical variables associated with titers in Japan.
Methods: We enrolled 365 healthcare workers (250 women, 115 men) whose three-month antibody
titers were analyzed in our previous study and whose blood samples were collected 183 ± 15 days
after the second dose. Participant characteristics, collected previously, were used. The relationships ofthese factors with antibody titers at six months and percentage changes in antibody titers from three
to six months were analyzed. Results: Median age was 44 years. Median antibody titer at six months
was 539 U/mL. Older participants had significantly lower antibody titers (20s, 752 U/mL; 60s–70s,
365 U/mL). In age-adjusted analysis, smoking was the only factor associated with lower antibody
titers. Median percentage change in antibody titers from three to six months was −29.4%. The only
factor significantly associated with the percentage change in Ab titers was not age or smoking, but
sex (women, −31.6%; men, −25.1%). Conclusion: The most important factors associated with lower
antibody titers at six months were age and smoking, as at three months, probably reflecting theireffect on peak antibody titers. However, the only factor significantly associated with the attenuationin Ab titers from three to six months was sex, which reduced the sex difference seen during the first
three months. Antibody titers may be affected by different factors at different time points.
Keywords: SARS-CoV-2; viral infection; clinical epidemiology

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article

1. Introduction

distributed under the terms and

The BNT162b2 vaccine (Pfizer/BioNTech), which was the first coronavirus disease2019 (COVID-19) mRNA vaccine to be administered to healthcare professionals in Japan,
starting in February 2021, is expected to help to prevent both the onset [1] and progressionof COVID-19. To obtain the maximum efficacy of vaccination with reduced adverse effects

conditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Vaccines 2021, 9, 1500

2 of 11and to optimize and individualize the vaccination regimen, we need to record the exact
chronological changes in real-world antibody (Ab) titers following BNT162b2 vaccinationand to investigate the relationships of recipients’ medical histories and demographic
characteristics with Ab titers.
Factors associated with the percentage change in Ab titers in the first few months after
the second of the two BNT162b2 doses could differ from those associated with the peak Abtiters immediately after the second of the two BNT162b2 doses. The median titer shortly
after the full schedule of this vaccine in Japan has been reported to be 2060 U/mL (interquartile range [IQR], 1250–2650 U/mL) [2], which is similar to the median titer reported
in Italy (1975 U/mL; IQR, 895–3455 U/mL) [3]. Various factors, including older age [2,4–6],
male sex [2], ethnicity [7], social condition [7], obesity [8,9], smoking habit [6,9], drinking habit [2], hypertension [9], cancer [10,11], and use of immunosuppressive drugs [2],have been reported to reduce the Ab titers obtained shortly after the second dose of the
BNT162b2 vaccine. In Japan, older age, male sex, and drinking habit are reported risk
factors for a lower peak value of the Ab titer [2].
In our previous cohort study of the serological Ab titers of 378 healthcare workers
at 3 months after the second dose of the BNT162b2 vaccine [12] in Tochigi prefecture,
Japan, these titers ranged from 3 to 5790 U/mL, and the median (IQR) Ab titer was
764 (423–1140) U/mL, which was much lower than the above-mentioned value obtained
shortly after the second inoculation. We thus determined that age and smoking status are
predictors of Ab titers three months after the second dose of the BNT162b2 vaccine in Japan.
However, the medium-term attenuation percentages of Ab titers several months after thesecond BNT162b2 dose and the relationship of these percentages with clinical backgroundand demographic characteristics are still not known.
Against this background, we analyzed Ab titers at six months and their percentagechange from three to six months after the second dose of the BNT162b2 vaccine andexplored the clinical variables associated with these parameters in Japan. To investigate theAb titers at three and six months, it is important to predict the period of effective Ab titers.
2. Methods
2.1. Population and Study Design
In this single-center prospective observational study, we recruited healthcare workers
whose blood samples were collected 183 ± 15 days after the second of two BNT162b2
vaccine inoculations (Pfizer/BioNTech) administered 3 weeks apart in February andMarch 2021 in the National Hospital Organization Utsunomiya National Hospital in
Tochigi prefecture, Japan.
Initially, we recruited 378 participants whose Ab titers 3 months after the second doseof the BNT162b2 COVID-19 vaccine were already analyzed in our previous study [12].
Their medical histories and demographic characteristics were recorded in that study using
a structured self-report questionnaire [12]. Vaccination was voluntary, and all vaccinatedindividuals were included as participants. However, we excluded 13 participants, namely,
5 who had already moved from our hospital, 2 who were absent, 3 who declined to participate, and 3 whose blood sampling confirmed the presence of Abs against the nucleocapsid
proteins for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Finally, we
enrolled 365 healthcare workers (250 women, 115 men). After informed consent was
obtained, we administered a questionnaire that covered personal information, such as
smoking, drinking, complications, and medication.
Blood samples collected 183 ± 15 days after the second inoculation were used tomeasure total Ab titers against the SARS-CoV-2 spike antigen using a commercially available electrochemiluminescence immunoassay (ECLIA) (Elecsys®Anti-SARS-CoV-2 RUO;
Roche Diagnostics) [13]. The relationships between Ab titers against the SARS-CoV-2 spike
antigen and clinical and lifestyle characteristics were analyzed. In age-adjusted analysis,
individual Ab titers were recalculated by subtracting the median Ab titer of the corresponding age group from an individual’s Ab titer. For example, an age-adjusted Ab titer for anVaccines 2021, 9, 1500

3 of 11individual in his/her 20s was calculated as follows: individual Ab titer—median Ab titerfor participants in their 20s.
In addition, we calculated the percentage change in Ab titers from 3 to 6 months after
the second dose of the BNT162b2 COVID-19 vaccine. Individual percentage changes in
Ab titers were calculated as follows: percentage change = ((Ab titer 6 months after the 2nd
dose − Ab titer 3 months after the 2nd dose [12])/Ab titer 3 months after the 2nd dose)× 100 (%). Then, the relationships between these percentage changes in Ab titers against
the SARS-CoV-2 spike antigen and clinical and lifestyle characteristics were analyzed.
The Ethics Committee of National Hospital Organization Utsunomiya National Hospital (No. 03-01; 19 April 2021) approved this study, and written informed consent was
obtained from all participants before enrollment.
2.2. Data Analysis
Nonparametric continuous data are expressed as the median with the IQR. Categorical
data are presented as absolute numbers and relative frequencies (n, %). To calculate
Spearman’s rank correlation coefficient and perform the Mann–Whitney U test, we used
Statistical Package for the Social Sciences (SPSS version 25). We considered p values lessthan 0.05 to be statistically significant.
3. Results
3.1. Study Population
In total, 365 healthcare workers (250 women, 115 men) were enrolled in this study.
Their baseline characteristics are summarized in Table 1. Briefly, the median (IQR) age andmean (SD) age of the participants was 44 (32–54) years and 43.9 (±13.5) years, respectively.
Nurses (n = 171) and physicians (n = 34) comprised 56.2% of the study population.
Table 1. Participants’ baseline characteristics (N = 365).
Variable

Total

Age, median (IQR), y

44 (32–54)

Sex (M/F), n

115/250

Body mass index, median (IQR),
kg/m2

22.4 (20.2–24.8)

Smoking (ever/never), n

149/216

Current smoker/
ex-smoker/unknown

90/45/14

Ab Titer at 6 Months,
Median (IQR), U/mL

Correlation
Coefficient ρ

p-Value−0.347<0.0001 #482 (305 to 865)/
572 (316 to 884)

0.1970 *−0.017406 (213 to 688)/
614 (405 to 957)
354 (174 to 564)/
436 (225 to 894) §§ex-smoker vs.
never-smoker
current smoker vs.
never-smoker

Brinkman Index §§ , §§§ , n

129

Drinking, n

228/134/3 §

544 (299 to 881)/
536 (348 to 868) §§Food

38/292/35 §Drug

37/293/35 §515 (307 to 925)/
544 (318 to 807) §§517 (287 to 759)/
549 (321 to 833) §§0.7446 #<0.0001 *0.0609 *0.0014 *<0.0001 *−0.1970.0258 #0.7697 *Allergy, n0.7697 *0.5858 *Vaccines 2021, 9, 1500

4 of 11

Table 1. Cont.
Variable
Allergic disease, nAllergic rhinitis
including pollinosis

Total

167/175/23 §Ab Titer at 6 Months,
Median (IQR), U/mL
546 (258 to 895)/523(320 to 792) §§582 (360 to 885)/
522 304 to 837) §§641(380 to 1005)/
522(303 to 815) §§Correlation
Coefficient ρ

p-Value

0.8242 *Bronchial asthma

43/299/23 §Skin allergy including
atopic dermatitis

47/295/23 §Diabetes mellitus, n

12/340/13 §331 (149 to 589)/
540 (318 to878) §§0.0512 *Hypertension, n27/325/13 §356 (195 to 566)/
553 (321to 872) §§0.0327 *Dyslipidemia, n18/334/13 §351 (205to 698)/
544 (317 to 877) §§0.0834 *Collagen disease, n

13/332/20 §303 (126 to 840)/
544 (324 to 843) §§0.2695 *0.5643 *0.0710 *IQR = interquartile range. *: Mann–Whitney U test. # : Spearman’s rank correlation coefficient test. § : yes/no/unknown are shown.
§§ : unknown is excluded. §§§ : ever-smoker only.

3.2. Distribution of Ab Titers against the SARS-CoV-2 Spike Sntigen 6 Sonths after the Second
Dose of the BNT162b2 COVID-19 Vaccine by Age and Sex
The median (IQR) Ab titer against the SARS-CoV-2 spike antigen 6 months after
vaccination with the BNT162b2 COVID-19 vaccine was 539 (309–872) U/mL. Older participants had significantly lower SARS-CoV-2 Ab titers (correlation coefficient ρ = −0.347)(Table 1 and Figure 1). Ab titers tended to decrease in both men and women as participants’age increased from their 20s to 70s. Median antibody titers of men in their 20s, 30s, 40s,
50s, and 60s–70s were 823, 648, 485, 335, and 432 U/mL, respectively, whereas median
Ab titers of women in their 20s, 30s, 40s, 50s, and 60s–70s were 748, 641, 560, 490, and318 U/mL, respectively.

Figure 1. Scatter plot of the distribution of Ab titers 6 months after the second vaccine dose according
to age and smoking status. Older participants had significantly lower Ab titers. Closed and open
circles show ever-smokers and never-smokers, respectively. Ever-smokers had lower Ab titers thannever-smokers.

Vaccines 2021, 9, 1500

5 of 113.3. Relationship between the Ab Titers against the SARS-CoV-2 Spike Antigen 6 Months after
Vaccination and Risk Factors
We first performed univariate analyses to identify factors associated with serum Ab
titers against the SARS-CoV-2 spike protein. The factors significantly associated with lower
Ab titers were older age, smoking, and hypertension (Table 1). Ever-smokers tended tohave lower Ab titers than never-smokers (Figure 1).
We also analyzed the risk factors for lower Ab titers after adjustment for age because
the prevalence of certain factors may differ according to age, such as hypertension. In the
age-adjusted analysis, individual Ab titers were recalculated by subtracting the median
Ab titer of the corresponding age group from an individual’s Ab titer. Median Ab titers ofparticipants in their 20s, 30s, 40s, 50s, and 60s–70s were 752, 642, 550, 418, and 365 U/mL,
respectively. Thus, the age-adjusted Ab titer for an individual in his/her 20s was calculated
as “individual Ab titer—752”. After age adjustment, the only factor significantly associatedwith lower Ab titers was smoking (Table 2). In terms of smoking, the age-adjusted median(IQR) Ab titers were −97 (−277 to 184) and 56 (−182 to 342) in ever-smokers and neversmokers, respectively.
Table 2. Age-adjusted data of median Ab titers (N = 365).
Variable

Ab Titer at 6 Months, Median (IQR),
U/mL

Male/female−15 (−246 to 241)/8 (−209 to 290)

Body mass index, median (IQR), kg/m2
Smoking (ever/never)Current smoker/ex-smoker

Correlation
Coefficient ρ

0.2851 *0.023

0.6647 #0.012<0.0001 *0.0255 *0.0203 *<0.0001 *0.8935 #−97 (−277 to 184)/56 (−182 to 342)
−205 (−320 to 7)/−72 (−264 to 256)ex-smoker vs. never-smoker
current smoker vs. never-smoker

Brinkman Index §§ , §§§p-Value

Drinking

3 (−242 to 296)/5 (−205 to 249) § , §§0.6475 *AllergyFood
Drug

18 (−250 to 235)/−2 (−223 to 256) § , §§−4 (−131 to 271)/−1 (−238 to 251) § , §§0.9604 *0.5858 *Allergic disease
Allergic rhinitis including pollinosis
Bronchial asthma
Skin allergy including atopic dermatitis−7 (−240 to 271)/−1 (−223 to 253) § , §§30 (−223 to 254)/−8 (−239 to 263) § , §§61 (−166 to 421)/−9 (−240 to 248) § , §§0.8473 *0.7390 *0.1638 *Diabetes mellitus−60 (−269 to 77)/0 (−225 to 272) § , §§0.4696 *Hypertension−69 (−247 to 183)/0 (−225 to 271) § , §§0.6409 *Dyslipidemia−56 (−220 to 259)/0 (−232 to 268) § , §§0.7392 *Collagen disease−227 (−368 to 290)/0 (−222 to 261) § , §§0.3021 *IQR = interquartile range. *: Mann–Whitney U test. # : Spearman’s rank correlation coefficient test. § : yes/no are shown. §§ : unknown is
excluded. §§§ : ever-smoker only.

3.4. Distribution of the Percentage Change in Ab Titers from 3 to 6 Months after the Second Doseof the BNT162b2 COVID-19 Vaccine by Age and Sex
The median (IQR) percentage change in Ab titers against the SARS-CoV-2 spike
antigen from three to six months after vaccination was −29.4% (−40.4% to −17.7%). No
significant correlation was observed between the percentage change in the anti-SARS-CoV2 Ab titer and age (correlation coefficient ρ = −0.028) (Table 3 and Figure 2A). Medianpercentage changes in Ab titers of men in their 20s, 30s, 40s, 50s, and 60s–70s were −24.3,−25.3, −24.0, −33.7, and −14.8 U/mL, respectively, whereas those of women in their 20s,
30s, 40s, 50s, and 60s–70s were −33.8, −31.5, −31.5, −30.0, and −32.6 U/mL, respectively.
Surprisingly, the only factor significantly associated with the percentage change in the Ab

Vaccines 2021, 9, 1500

6 of 11titer was not age or smoking, but sex (Table 3). In terms of sex, median (IQR) percentagechanges in Ab titers were −31.6% (−42.0% to −20.2%) and −25.1% (−36.8% to −12.0%) in
women and men, respectively (Table 3), suggesting that Ab titers decrease faster in womenthan in men.
An additional analysis was performed (Table 4) because smoking was an importantrisk factor at both three and six months after vaccination and the smoking percentages
differed between men and women. For age-adjusted median Ab titers, no significant
sex differences were observed in the ever-smoker and never-smoker groups. However,both the male and female groups showed significant differences by smoking status inage-adjusted median Ab titers. On the other hand, no significant differences in the medianpercentage change in Ab titers by smoking status were observed in the male and female
groups. However, both the ever-smoker and never-smoker groups showed significant sex
differences in the median percentage change in Ab titers.
The relationship between the percentage changes in Ab titers and the Ab titer 6 months
after vaccination was analyzed (Figure 2B). A significant positive correlation was observed,
indicating that participants with a lower Ab titer had greater attenuation of the Ab titer.
Table 3. Median percentage change in Ab titers from 3 months to 6 months (N = 365).
Variable

Percentage Change in Ab Titers,
Median (IQR), % §AgeMale/female

p-Value−0.0280.5876 #−25.1 (−36.8 to −12.0)/−31.6 (−42.0 to −20.2)

Body mass index, median (IQR), kg/m2
Smoking (ever/never)Current smoker/ex-smoker

Correlation
Coefficient ρ

0.0005 *0.025

0.6274 #−0.0340.3051 *0.3853 *0.2914 *0.8809 *0.6990 #−28.4 (−39.7 to −15.5)/−30.3 (−40.7 to −19.0)−31.7 (−40.6 to −18.3)/−27.4 (−40.1 to −16.1) §§ex-smoker vs. never-smoker
current smoker vs. never-smoker

Brinkman Index §§ , §§§Drinking−29.1(−41.0 to −17.8)/−30.5(−39.1 to −18.1) § , §§0.777 *

AllergyFood
Drug−29.4 (−37.1 to −16.5)/−29.4 (−39.9 to −18.6) § , §§−28.8 (−40.7 to −22.6)/−29.7 (−39.3 to −18.3) § , §§0.7821 *0.7469 *Allergic disease
Allergic rhinitis including pollinosis
Bronchial asthma
Skin allergy including atopic dermatitis−29.2 (−40.9 to −18.1)/−29.6 (−39.6 to −18.0) § , §§−31.7 (−40.5 to −19.4)/−29.1 (−40.5 to −17.6) § , §§−30.3 (−44.6 to −25.1)/−29.1 (−39.7 to −17.6) § , §§0.8606 *0.5521 *0.1677 *Diabetes mellitus−27.6 (−32.8 to −11.4)/−29.4 (−40.5 to −18.0) § , §§0.3617 *Hypertension−24.0 (−32.1 to −12.3)/−29.7 (−40.8 to −18.0) § , §§0.0679 *Dyslipidemia−31.7 (−39.2 to −22.8)/−29.3 (−40.5 to −17.7) § , §§0.7178 *Collagen disease−27.0 (−48.6 to −19.1)/−29.6 (−40.3 to −17.9) § , §§0.889 *

IQR = interquartile range. *: Mann–Whitney U test. # : Spearman’s rank correlation coefficient test. § : yes/no are shown. §§ : unknown is
excluded. §§§ : ever-smoker only.

Vaccines 2021, 9, 1500

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Figure 2. Scatter plot of the distribution of the percentage change in Ab titers from 3 to 6 months after
the second dose of the vaccine according to sex. The relationship between the percentage change after
the vaccination and age is shown in (A), and the relationship between the percentage change and theAb titer 6 months after the vaccination is shown in (B). No significant correlation was observed in(A), and age did not affect the attenuation of the Ab titers from 3 to 6 months after the vaccination.
However, a significant correlation was observed in (B). Closed and open circles and continuous andbroken lines show men and women, respectively.

Vaccines 2021, 9, 1500

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Table 4. Age-adjusted median Ab titers and percentage changes in the relationship between sex and smoking.

Variable

Male n,Ab, Median (IQR), U/mL
Percentage Change, Median (IQR),%

Female n,
Ab, Median (IQR), U/mL
Percentage Change, Median (IQR),%

Ever-smokers

70−120 (−266 to 152)−25.9% (−38.0 to −12.0)

79−68 (−278 to 193)
−30.5% (−42.0 to −18.7)

0.5709 *0.0400 *Never-smokers

45
115 (−181 to 519)
−24.0% (−34.0 to −12.1)

171
46 (−181 to 332)
−31.7% (−42.1 to −20.9)

0.4700 *0.0050 *0.0040 *0.7613 *0.0120 *0.7018 *p-value for
Ab titer
Percentage change

p-Value for
Ab Titer
Percentage Change

IQR = interquartile range. *: Mann–Whitney U test.

4. Discussion
To our knowledge, this is the first study to report real-world Ab titers against the
SARS-CoV-2 spike antigen at six months and the percentage changes in Ab titers from three
to six months after vaccination and to identify the factors associated with these parametersfrom a comprehensive range of clinical and lifestyle characteristics in Japan. Few studies
have examined this issue [14,15]. Four important findings were obtained. First, in a cohort
with a median (IQR) age of 44 (32–54) years, the median Ab titer six months after thesecond dose was 539 U/mL. Older participants had significantly lower Ab titers, with an
Ab titer about half that of those in their 20s. Second, in an age-adjusted analysis, the onlyrisk factor for a lower Ab titer was smoking. Third, the median percentage change in the
Ab titer from three to six months after vaccination was −29.4%. Surprisingly, the only
factor significantly associated with attenuation of the Ab titers was not age or smoking, but
sex. The median percentage changes in Ab titers were −31.6% and −25.1% in women andmen, respectively. Fourth, both male and female participants with a lower Ab titer showed
greater attenuation of the Ab titer. This means that the percentage change in Ab titers may
be affected by individual differences.
In terms of the first and second findings, the most important factors associated witha low Ab titer six months after the second dose were still age and smoking habit, as seen
three months after the second dose, probably reflecting their effect on peak Ab titers.
Interestingly, a reversal of the sex difference in Ab titers seen three months after thesecond dose was observed at six months. In our previous study, the median Ab titerwas 764 U/mL three months after the second dose [12]. The median Ab titers were 942
and 1095 U/mL in men and women in their 20s, respectively, but 490 and 519 U/mL in
men and women in their 60s–70s, respectively. In the age-adjusted analysis, the only risk
factors for a lower Ab titer three months after the second dose [12] were male sex andsmoking. However, we concluded that the sex difference may have arisen from the sex
difference in the smoking percentage, rather than from biological sex differences. We foundseveral studies reporting an association between smoking and lower Ab titers against
both influenza virus [16] and hepatitis B virus [17] after vaccination, although the actual
mechanisms are poorly understood.
Regarding the third finding, the only factor significantly associated with the attenuation in Ab titers from three to six months after the second dose was not age or smoking, butsex, which seemingly resulted in a reduction of the sex difference in Ab titers seen threemonths after the second dose. This suggests that Ab titers decrease faster in women thanin men.
Concerning the fourth finding, we hypothesized that participants with a higher Abtiter would show greater attenuation of Ab titers because the percentage change from three
to six months and the Ab titer three months after vaccination were significantly higher inVaccines 2021, 9, 1500

9 of 11

women than in men. However, a significant positive correlation was observed between thepercentage change in Ab titers and the Ab titer six months after vaccination. The Ab titerat six months after vaccination was associated with the percentage attenuation from three
to six months, and participants with a lower Ab titer showed greater attenuation of thetiter. We could not find other risk factors, such as age and comorbidities, beyond the above.
Although we concluded that the attenuation was affected by individual differences, wewill continue to define the risk factors through additional study.
The factors identified to affect Ab titers vary by the time elapsed since vaccination.
For peak Ab titers 2–5 weeks after vaccination, the reported risk factors were sex, age,
and alcohol intake. Three months after vaccination, age and smoking were risk factors
in our study, and the effects of sex and alcohol intake were not observed. Six months
after vaccination, age and smoking were still risk factors in our study. However, thesefactors did not affect the attenuation from three to six months. This means that these factors
will strongly affect the Ab titer within three months after vaccination and that the effects
will persist six months after vaccination. From three to six months after vaccination, sex
and lower Ab titer were risk factors. According to the above results, smoking is the mostimportant factor that could be avoided to maintain a higher Ab titer. Thus, it appears thatdifferent factors affect the Ab titer at different time points.
Some limitations and possible sources of bias in this study include the following. First,
the participants were limited in number and were all healthcare workers vaccinated ata single national hospital in Tochigi prefecture. Therefore, the results obtained in thisstudy might not be generalizable on a wide scale, or even within Japan. Second, we
excluded participants with Abs against nucleocapsid proteins on the presumption thatthey had previously been infected with COVID-19. However, some of these patients had
slight increases in Ab titers against the spike protein but became negative for Ab titers
against nucleocapsid proteins. One possibility is that they had not been infected with
COVID-19 and that the Ab titers against the spike protein had been increased by individual
differences. The other possibility is that the Ab titers against nucleocapsid proteins might
have not increased due to a small viral load of SARS-CoV-2, even though they may still
have been infected with COVID-19. To determine the chance of exposure to a small amountof SARS-CoV-2 virus, we analyzed Ab titers in participants who worked in the COVID-19
ward (data not shown). However, their Ab titers were not increased. If the excluded
participants were by chance exposed to a small amount of SARS-CoV-2 virus, it may havebeen through daily life and not the COVID-19 ward. In addition, five participants had
Ab titers exceeding 3000 U/mL and/or a greater than 80% increase in Ab titers against
the spike protein, despite negative Abs against nucleocapsid proteins. They may havebeen infected with COVID-19, considering that a participant with an Ab titer against spike
proteins exceeding 5000 U/mL and without Ab titers against nucleocapsid proteins threemonths after vaccination had positive Ab titers against nucleocapsid proteins at six months.
The participant may have been infected with COVID-19 three months after vaccinationand the Abs against nucleocapsid proteins may have increased at a late stage due to theinfection. However, we do not have evidence that the above five participants were infectedwith COVID-19 and we did not exclude them from our analysis.
In conclusion, the most important factors associated with a low Ab titer six months
after the second dose were age and smoking habit, as seen three months after the second
dose, probably reflecting the effects of these factors on peak Ab titers. However, theattenuation of Ab titers from three to six months after the second BNT162b2 dose depended
not on age or smoking, but on sex. A significant attenuation of Ab titers from three to six
months was solely observed in women and resulted in a reduction of the sex differencein the Ab titer seen at three months. Further studies of the associations between Ab titers
and the comprehensive medical histories of individuals are needed to establish a more
personalized approach to vaccination involving earlier boosters, different schedules, ordifferent types of vaccines.

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Author Contributions: Conceptualization, M.S., K.S., Y.N. (Yushi Nomura) and Y.N. (Yosikazu
Nakamura); methodology and software, Y.N. (Yushi Nomura), K.S., M.S. and Y.N. (Yosikazu
Nakamura); validation, K.S., M.S. and Y.N. (Yosikazu Nakamura); formal analysis and investigation, K.S., Y.N. (Yushi Nomura), M.S. and Y.N. (Yosikazu Nakamura); resources, T.T., K.S., N.M.,
Y.N. (Yushi Nomura), M.S., O.K. and R.K.; data curation, Y.N. (Yushi Nomura), K.S. and M.S.;
writing—original draft preparation, M.S., K.S., Y.N. (Yushi Nomura) and Y.N. (Yosikazu Nakamura);
writing—review and editing, T.T., N.M., S.N., K.H., O.K. and R.K.; visualization, K.S., Y.N. (Yushi
Nomura) and M.S.; supervision, M.S., K.S., T.T., S.N. and K.H.; project administration, K.S. andT.T.; funding acquisition, T.T. and K.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee of National Hospital Organization
Utsunomiya National Hospital (No. 03-01, 19 April 2021).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Acknowledgments: The authors thank all staff at National Hospital Organization Utsunomiya
National Hospital for providing support during sample collection. We would like to thank Miwa
Okada, Junko Shibayama, Yuko Tajima, Mami Ochiai, Midori Takahashi, Hiroko Ueno, Natsuka
Suzuki, and Yoshino Iwaya for providing support during data analysis.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
COVID-19: coronavirus disease 2019; Ab: antibody; IQR: interquartile range;
SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

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