Article

Age and Smoking Predict Antibody Titres at 3 Months after theSecond Dose of the BNT162b2 COVID-19 Vaccine
Yushi Nomura 1,2 , Michiru Sawahata 1,3, * , Yosikazu Nakamura 4 , Momoko Kurihara 1 , Ryousuke Koike 1,2 ,Otohiro Katsube 1 , Koichi Hagiwara 3 , Seiji Niho 2 , Norihiro Masuda 5 , Takaaki Tanaka 6 and Kumiya Sugiyama 1,7
1

2

3

4
5

6

7




*Department of Respiratory Medicine and Clinical Immunology, National Hospital Organization Utsunomiya
National Hospital, Utsunomiya 329-1193, Japan; uc.nomura@gmail.com (Y.N.);
momoko1005kurihara@gmail.com (M.K.); 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
Division of Pulmonary Medicine, Department of 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
Department of Respiratory Medicine and Clinical Immunology, Dokkyo Medical University Saitama Medical Center,
Shimotsuga 321-0293, Japan
Correspondence: sawahata@jichi.ac.jp; Tel.: +81-285587350

Citation: Nomura, Y.; Sawahata, M.;
Nakamura, Y.; Kurihara, M.; Koike,
R.; Katsube, O.; Hagiwara, K.; Niho,
S.; Masuda, N.; Tanaka, T.; et al. Ageand Smoking Predict Antibody Titresat 3 Months after the Second Dose of
the BNT162b2 COVID-19 Vaccine.
Vaccines 2021, 9, 1042. https://doi.org/10.3390/vaccines9091042
Academic Editors: Ger Rijkers
and Jean-Luc Murk
Received: 6 September 2021
Accepted: 16 September 2021
Published: 18 September 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in

Abstract: Objective: We aimed to determine antibody (Ab) titres 3 months after the second doseof the BNT162b2 coronavirus disease-2019 (COVID-19) vaccine and to explore clinical variables
predicting these titres in Japan. Methods: We enrolled 378 healthcare workers (255 women, 123 men)whose blood samples were collected 91 ± 15 days after the second of two inoculations of the
BNT162b2 COVID-19 mRNA vaccine (Pfizer/BioNTech) given 3 weeks apart. Medical histories
and demographic characteristics were recorded using a structured self-reported questionnaire. The
relationships between Ab titres and these factors were analysed. Results: Median age (interquartile
range (IQR)) of the participants was 44 (32–54) years. Median Ab titre (IQR) against the severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen was 764 (423–1140) U/mL. Olderparticipants had significantly lower Ab titres; median (IQR) Ab titres were 942 (675–1390) and 1095
(741–1613) U/mL in men and women in their 20s, respectively, but 490 (297–571) and 519 (285–761)
U/mL in men and women in their 60–70s, respectively. In the age-adjusted analysis, the only risk
factors for lower Ab titres were male sex and smoking. However, the sex difference may have arisen
from the sex difference in smoking rate. Moreover, Ab titres were significantly lower in current
smokers than in ex-smokers. Conclusions: The most important factors associated with low Ab titres
were age and smoking habit. In particular, current smoking status caused lower Ab titres, andsmoking cessation before vaccination may improve the individual efficacy of the BNT162b2 vaccine.

published maps and institutional affiliations.

Keywords: SARS-CoV-2; viral infection; clinical epidemiology

Copyright: © 2021 by the authors.

1. Introduction

Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

In Japan, the BNT162b2 vaccine (Pfizer/BioNTech) was selected as the first coronavirus
disease-2019 (COVID-19) mRNA vaccine to be administered to healthcare professionals,
starting in February 2021. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
has an enveloped, single, positive-stranded RNA genome that encodes four major viralstructural proteins, namely nucleocapsid (N), spike (S), envelope (E) and membrane (M)proteins; the latter three proteins are found in its membrane. The spike protein guides viral

Vaccines 2021, 9, 1042. https://doi.org/10.3390/vaccines9091042

https://www.mdpi.com/journal/vaccines

Vaccines 2021, 9, 1042

2 of 9entry into host cells [1] by binding to ACE2 (angiotensin-converting enzyme 2), the main
virus receptor, which is widely expressed on epithelial cells and macrophages [1–3] and isthus an ideal target for mRNA vaccine development [3].
Because efficacy in clinical trials and effectiveness in the community depend on the proportions of SARS-CoV-2 variants spreading in a given area, immunogenicity has attracted
increasing attention as an individual index for the efficacy of COVID-19 mRNA vaccines.
Humoral immunity plays major roles in protecting against and surviving SARS-CoV-2
infection [4,5], and neutralising antibodies (Abs) are correlated with protection against
several viruses, including SARS-CoV-2 [6–9]. However, few studies have investigated realworld Ab titres following vaccination with BNT162b2; focus has been placed mostly on the
Ab status shortly after vaccination, which provides information for predicting long-term
effectiveness [10–12]. These studies focused on short-term Ab titre data and demonstrated
that lower Ab titres were associated with older age [10–13], male sex [10], ethnicity [14],social condition [14], obesity [15,16], smoking habit [13,16], drinking habit [10], hypertension [16], cancer [17,18], use of immunosuppressive drugs [10] and a longer period of time
elapsed after vaccine inoculation [10,16].
Therefore, in our preliminary study, we reported the medium-term data on Ab
titres against the SARS-CoV-2 spike antigen produced in response to this mRNA vaccine in the Japanese population. Our results showed that the average peak titre of2031.7 ± 692.0 U/mL in six healthcare workers in their 50s and 60s had markedly decreased to 513.3 ± 261.7 U/mL by 15 weeks after the second inoculation (data not shown).
Here, we aimed to determine Ab titres against the SARS-CoV-2 spike antigen 3 months
after the second dose of the BNT162b2 vaccine in 378 healthcare workers and to explore
the factors associated with these Ab titres across a comprehensive range of clinical andlifestyle characteristics in Japan.
2. Methods
2.1. Population and Study Design
In this single-centre, prospective, observational study, we recruited healthcare workers whose blood (serum) samples were collected 91 ± 15 days after the second of two
BNT162b2 vaccine inoculations (Pfizer/BioNTech) administered 3 weeks apart in February–
March 2021 in National Hospital Organization Utsunomiya National Hospital in Tochigi
prefecture, Japan.
Initially, 381 participants were recruited, but we excluded 1 participant who received
only the first dose of the BNT162b2 vaccine and 2 participants whose blood sampling
confirmed the presence of Abs against SARS-CoV-2 (nucleocapsid proteins) prior to vaccination. Finally, we enrolled 378 healthcare workers (255 women, 123 men), including the 6from our aforementioned preliminary study, and their medical histories and demographic
characteristics were recorded using a structured self-reported questionnaire.
Blood samples collected 91 ± 15 days after the second inoculation were used immediately after sampling to measure total Ab titres against the SARS-CoV-2 spike antigen, using
a commercially available electrochemiluminescence immunoassay (ECLIA) (Elecsys® AntiSARS-CoV-2 RUO; Roche Diagnostics) [19]. The relationships between Ab titres against
the SARS-CoV-2 spike antigen and clinical and lifestyle characteristics were analysed.
This study was approved by the Ethics Committee of National Hospital Organization
Utsunomiya National Hospital (No. 03-01, 19 April 2021). Written informed consent was
obtained from all study participants before enrolment.
2.2. Data Analysis
Nonparametric continuous data are expressed as the median with interquartile range(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).

Vaccines 2021, 9, 1042

3 of 9

3. Results
3.1. Study Population
In total, 378 healthcare workers (255 women, 123 men) were enrolled in this study.
Their baseline characteristics are summarised in Table 1. Briefly, median age (IQR) of theparticipants was 44 (32–54) years. Nurses (n = 177) and physicians (n = 38) comprised
56.9% of the study population.
Table 1. Baseline characteristics of participants (N = 378).
Variable

Total

Age, median (IQR), y

44 (32–54)

Sex (Male/female), n

123/255

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

Antibody Titre,Median (IQR), U/mL

38 (10.1)
177 (46.8)
34 (9.0)
10 (2.6)
7 (1.9)p-Value−0.386<0.0001 #0.0193 *

652 (388–1025)/825 (458–1210)−0.00722.4 (20.3–24.8)

Occupation, n (%)
Physician
Nurse
Clerical work
Pharmacist
Clinical laboratory
technician
Radiologist
Rehabilitation staff
Caregiver
OthersCorrelation
Coefficient ρ

690 (550–952)
875 (500–1240)
669 (424–1033)

0.8975 #0.1669 (vs. Nurse) *0.1015 (vs. Clerical) *0.7181 (vs. Physician) *7 (1.9)18 (4.8)
11 (2.9)76 (20.1)

Smoking (ever/never), n
Brinkman index, n<0.0001 *0.0062 #154/224
134 §§§

528 (320–910)/919 (587–1373)

Drinking, n

235/140/3 §

758 (418–1175)/823 (471–1133) §§0.7244 *Allergy, n
Food
Drug

40/302/36 §38/303/37 §

745 (374–1208)/757 (435–1098) §§757 (439–1103)/758 (434–1130) §§0.8223 *0.7988 *Allergic disease, nAllergic rhinitis including pollinosis
Bronchial asthma
Skin allergy including atopic dermatitis

178/176/24 §46/308/24 §49/305/24 §

780 (385–1138)/696 (446–1103) §§876 (449–1135)/742 (418–1133) §§924 (582–1410)/733 (415–1080) §§0.7839 *0.5095 *0.0413 *Diabetes mellitus, n

12/353/13 §382 (211–741)/768 (436–1150) §§0.0189 *Hypertension, n27/338/13 §521 (285–869)/777 (443–1158) §§0.0120 *Dyslipidaemia, n18/347/13 §569 (340–878)/768 (434–1155) §§0.0741 *Collagen disease, n

13/345/20 §548 (256–1030)/766 (435–1140) §§0.3170 *−0.237

IQR = interquartile range. *: Mann–Whitney U test. # : Spearman’s rank correlation coefficient test. § : yes/no/unknown are shown.
§§ : Antibody titres are shown for yes/no, excluding unknown. §§§ : ever smoking only.

3.2. Distribution of Ab Titres against SARS-CoV-2 Spike Antigen 3 Months after Vaccination
According to Age and Sex
Median Ab titre (IQR) against the SARS-CoV-2 spike antigen was 764 (423–1140) U/mL
(Table 1). Older participants were found to have significantly lower SARS-CoV-2 Ab titres(correlation coefficient ρ = −0.386, p < 0.0001) (Figure 1). The Ab titres in both men andwomen tended to decrease with an increase in age from 20s to 70s (Figure 2). Median (IQR)
Ab titres of men in their 20s, 30s, 40s, 50s and 60s–70s were 942 (675–1390), 821 (484–1115),
710 (393–938), 449 (289–861) and 490 (297–571) U/mL, respectively. Median (IQR) Ab titresof women in their 20s, 30s, 40s, 50s and 60s–70s were 1095 (741–1613), 991 (613–1410),
827 (501–1150), 685 (377–1035) and 519 (285–761) U/mL, respectively. Men in each age
group tended to have lower median Ab titres than those of women in the same age group,
although the differences were not significant (Figure 2).

Vaccines 2021, 9, 1042

Ab titres (correlation coefficient ρ = −0.386, p < 0.0001) (Figure 1). The Ab titres in both men
and women tended to decrease with an increase in age from 20s to 70s (Figure 2). Median(IQR) Ab titres of men in their 20s, 30s, 40s, 50s and 60s–70s were 942 (675–1390), 821 (484–1115), 710 (393–938), 449 (289–861) and 490 (297–571) U/mL, respectively. Median (IQR)
Ab titres of women in their 20s, 30s, 40s, 50s and 60s–70s were 1095 (741–1613), 991 (613–1410), 827 (501–1150), 685 (377–1035) and 519 (285–761) U/mL, respectively. Men in eachage group tended to have lower median Ab titres than those of women in the same agegroup, although the differences were not significant (Figure 2).

4 of 9Figure 1.1.Scatterplotof thedistributionof antibody
titres according
to age. Medianantibody
titre antibody titre
FigureScatterplotof thedistributionof antibody
titres according
to age.
Median(IQR) against the SARS-CoV-2 spike antigen was 764 (423–1140) U/mL. Older participants had sigVaccines 2021, 9, x FOR PEER REVIEW
of 10
(IQR)
againstthe SARS-CoV-2
spike
was
764 (423–1140)
had
nificantly
lower SARS-CoV-2
antibody
titres antigen(correlation
coefficientρ = −0.386).U/mL. Older 5participantssignificantly lower SARS-CoV-2 antibody titres (correlation coefficient ρ = −0.386).

Figure 2. Distribution of antibody titres according to age group and sex. Antibody titres in both men
Figure2. Distribution of antibody titres according to age group and sex. Antibody titres in both men
and women tended to decrease as participants’ ages increased from their 20s to 70s. Median (IQR)
and
women
as participants’ages increased
from
their
Median (IQR)
antibody titrestendedof men to
in decrease
their 20s, 30s,
40s, 50s and 60s–70s
were 942, 821,
710,
449 20s
and to49070s.
U/mL,
antibody
titres
of
men
in
their
20s,
30s,
40s,
50s
and
60s–70s
were
942,
821,
710,
449
and
respectively. Median (IQR) antibody titres of women in their 20s, 30s, 40s, 50s and 60s–70s were 490 U/mL,
1095, 991, 827, 685
and 519
U/mL,
respectively.
Meneach ageingroupto have
medianrespectively.
Median(IQR)
antibody
titresofinwomen
theirtended
20s, 30s,
40s,lower
50s and60s–70s were
antibody titres than women in the same age group. * p < 0.05, ** p < 0.01.
1095, 991, 827, 685 and 519 U/mL, respectively. Men in each age group tended to have lower medianantibody
titres than
womenin theagainstsame SARS-CoV-2
age group. * Spikep < 0.05,** p <
0.01. after Vaccina3.3. Relationshipbetween
Ab Titres
Antigen3 Months

tion and Risk Factors
3.3.
Relationship between Ab Titres against SARS-CoV-2 Spike Antigen 3 Months after
We firstandperformed
univariate analyses to identify factors associated with serum Ab
Vaccination
Risk Factors
titres against the SARS-CoV-2 spike protein. The factors significantly associated with a
We first performed univariate analyses to identify factors associated with serum Ab
lower Ab titre were older age, male sex, smoking, skin allergy (including atopic dermatititres
againstthe SARS-CoV-2
spike(Table
protein.
tis), diabetes
mellitusand hypertension1). The factors significantly associated with a
We also analysed the risk factors for lower Ab titres after adjustment for age, because
the prevalence of the factors may differ according to age, such as hypertension. In the ageadjusted analysis, individual Ab titres were recalculated by means of subtraction of themedian Ab titres according to corresponding age groups. Median Ab titres of participantsin their 20s, 30s, 40s, 50s and 60s-70s were 1050, 931, 779, 619 and 491 U/mL, respectively.

Vaccines 2021, 9, 1042

5 of 9lower Ab titre were older age, male sex, smoking, skin allergy (including atopic dermatitis),diabetes mellitus and hypertension (Table 1).
We also analysed the risk factors for lower Ab titres after adjustment for age, because
the prevalence of the factors may differ according to age, such as hypertension. In the
age-adjusted analysis, individual Ab titres were recalculated by means of subtraction of themedian Ab titres according to corresponding age groups. Median Ab titres of participantsin their 20s, 30s, 40s, 50s and 60s-70s were 1050, 931, 779, 619 and 491 U/mL, respectively.
For example, an age-adjusted Ab titre in a participant in their 20s was calculated asfollows: “an individual Ab titre—1050”. After age adjustment, the only factors significantly
associated with lower Ab titres were male sex and smoking (Table 2). In terms of smoking,age-adjusted median Ab titres (IQR) were −174 (−378 to 145) and 90 (−174 to 512) in eversmokers and never smokers, respectively.
Table 2. Age-adjusted data of median antibody titres (N = 378).
Total

Antibody Titre,Median (IQR) (U/mL)

Male/female

123/255−69 (−373 to 216)/55 (−266 to 426)

0.0170 *Occupation, n (%)
Physician
Nurse
Clerical work

38 (10.1)
177 (46.8)
34 (9.0)31 (−263 to 144)
46 (−335 to 440)
−91 (−309 to 301)

0.6069 (vs. Nurse) *0.5018 (vs. Clerical) *0.7479 (vs. Physician) *Smoking (ever/never), n
Brinkman index

154/224
134 §§§−174 (−378 to 145)/90 (−174 to 512)

Drinking, n

235/140/3 §−18 (−329 to 371)/32 (−267 to 318) §§0.4955 *Allergy, n
Food
Drug

40/302/36 §38/303/37 §36 (−393 to 357)/−7 (−296 to 319) §§114 (−165 to 317)/−15 (−309 to 320) §§0.7336 *0.3018 *Allergic disease, nAllergic rhinitis including pollinosis
Bronchial asthma
Skin allergy including atopic dermatitis

178/176/24 §46/308/24 §49/305/24 §10 (−334 to 331)/−19 (−289 to 320) §§93 (−324 to 329)/−23 (−303 to 321) §§129 (−257 to 522)/−28 (−308 to 299) §§0.8541 *0.6102 *0.0826 *Diabetes mellitus, n

12/353/13 §−169 (−315 to 82)/0 (−300 to 342) §§0.3105 *Hypertension, n27/338/13 §−88 (−361 to 284)/0 (−286 to 338) §§0.3771 *Dyslipidaemia, n18/347/13 §24 (−280 to 114)/−3 (−300 to 340) §§0.7484 *Collagen disease, n

13/345/20 §−90 (−516 to 252)/0 (−299 to 334) §§0.3085 *Variable

Correlation
Coefficient ρ−0.003

p-Value<0.0001 *0.97010 #IQR = interquartile range. *: Mann–Whitney U test. # : Spearman’s rank correlation coefficient test. § : yes/no/unknown are shown.
§§ : Antibody titres are shown for yes/no, excluding unknown. §§§ : ever smoking only.

To analyse the observed sex difference, we divided the participants into ever smokers
and never smokers. In both the male and female groups, age-adjusted median Ab titres
were significantly lower in ever smokers than in never smokers; age-adjusted median Ab
titres (IQR) in men were −246 (−398 to 65) and 49 (−186 to 621) in ever smokers and neversmokers, respectively, while those in women were −140 (−304 to 217) and 95 (−151 to 503)
in ever smokers and never smokers, respectively. However, in both the ever smoker andnever smoker groups, no significant sex differences in age-adjusted median Ab titres wereobserved (Table 3). Given that the smoking rates in the male and female groups were 61.0%
and 31.0%, respectively, these results suggest that the sex difference in Ab titres strongly
reflects sex differences in smoking, rather than biological sex differences. In addition, Ab
titres were significantly lower in current smokers than in ex-smokers (Table 4). These
results suggest that smoking cessation will reduce the risk of a lower Ab titre.

Vaccines 2021, 9, 1042

6 of 9

Table 3. Age-adjusted median antibody titres in ever smokers vs. never smokers.
Male, Antibody Titre,Median (IQR), U/mL

Female, Antibody Titre,Median (IQR), U/mL

p-Value between Sexes

Ever smokers (men, 75; women, 79)−246 (−398 to 65)−140 (−304 to 217)

0.1175 *Never smokers (men, 48; women, 176)

49 (−186 to 621)

95 (−151 to 503)

0.9970 *p-value between ever and never smokers

0.0007 *0.0023 *IQR = interquartile range. *: Mann–Whitney U test.

Table 4. Age-adjusted median antibody titres in ever smokers: current smokers vs. ex-smokers.
Antibody Titre,Median (IQR), U/mL

p-Value(vs. Never Smokers)

Current smokers (n = 49)−271 (−475 to 33)<0.0001 *Ex-smokers (n = 91)−162 (−332 to 285)

0.0019 *P-value (current vs. ex-smokers)

0.0188 *IQR = interquartile range. *: Mann–Whitney U test.

4. Discussion
To our knowledge, this is the first study to report real-world Ab titres against the SARSCoV-2 spike antigen at 3 months after vaccination and to identify the factors associatedwith these Ab titres across a comprehensive range of clinical and lifestyle characteristics inJapan. Three important findings were obtained. First, median age (IQR) of the participants
was 44 (32–54) years, median Ab titre (IQR) against the SARS-CoV-2 spike antigen was
764 (423–1140) U/mL, and older participants had significantly lower Ab titres, with median(IQR) Ab titres of 942 (675–1390) and 1095 (741–1613) U/mL in men and women in their
20s, respectively, but 490 (297–571) and 519 (285–761) U/mL in men and women in their60s–70s, respectively. Second, in the age-adjusted analysis, the only risk factors for lower
Ab titres were male sex and smoking. However, the sex difference may have arisen from thesex difference in smoking rate. Other health complications, including diabetes mellitus andallergic diseases, were not correlated with lower Ab titres after age adjustment. Third, Ab
titres were significantly lower in current smokers than in ex-smokers. Smoking cessation
can thus be expected to reduce the risk of lower Ab titres.
Because the efficacy of COVID-19 mRNA vaccines in clinical trials and their effectiveness in the community depend on the proportions of SARS-CoV-2 variants spreading ina given area, immunogenicity has attracted increasing attention as an individual index
for the efficacy of these vaccines. Neutralising Abs are correlated with protection against
SARS-CoV-2 [5], but only a few studies have investigated real-world Ab titres following vaccination with BNT162b2, focusing instead on Ab status shortly after vaccination.
Those studies demonstrated that lower Ab titres may be caused by older age [10–13], malesex [10], ethnicity [14], social condition [14], obesity [15,16], smoking habit [13,16], drinking
habit [10], hypertension [16], cancer [17,18], use of immunosuppressive drugs [10] anda longer period of time elapsed after vaccine inoculation [10,16]. Medium-term data ofserological Ab titres in response to the BNT162b2 vaccine are urgently needed, as are theclinical and lifestyle factors predicting these titres.
Concerning our first main finding, the median titre in individuals shortly after afull vaccination schedule of this vaccine in Japan was reported to be 2060 U/mL (IQR,
1250–2650) [10], which is similar to the median titre reported from Italy (1975 U/mL;
IQR, 895–3455) [20]. However, Ab titres against the SARS-CoV-2 spike antigen following
vaccination cannot currently predict the likelihood of developing COVID-19. Our Ab titres
3 months after the second inoculation ranged from 3 to 5790 U/mL, and the median Ab
titre (IQR) was 764 (423–1140) U/mL, which was much lower than the above-mentioned
value obtained shortly after the second inoculation, reflecting the reported association
between a longer period of time elapsed since the second inoculation and lower Ab titres.

Vaccines 2021, 9, 1042

7 of 9Moreover, older participants had significantly lower SARS-CoV-2 Ab titres. Indeed, in
our preliminary study with six participants in their 50s and 60s, the average peak titreof 2031.7 ± 692.0 U/mL had markedly decreased to 513.3 ± 261.7 U/mL by 15 weeks
after the second inoculation (data not shown). Two of the six subjects had Ab titres of
220.0 U/mL, which is a similar level to that observed immediately before the second
inoculation, suggesting that the third vaccination with COVID-19 mRNA vaccine mayneed to be added, at least in part, for people over 50 years old in Japan. Although sufficient
clinical efficacy of the BNT162b2 vaccine against most of the conventional SARS-CoV-2
variants was observed even after 6 months without Ab titre data [21], the clinical efficacy
of a single inoculation with this vaccine against the SARS-CoV-2 Delta variant has beenreported to be only 30.7% [22].
Concerning our second main finding, various reports have demonstrated that our participants receiving immunosuppressive drugs also seemed to have low Ab titres, including
a man in his 30s taking infliximab [13] with a titre of 9.0 U/mL, a woman in her 40s taking
etanercept with 258 U/mL and a woman in her 30s taking etanercept with 415 U/mL. In
our study, smoking was the most impactful factor, and the sex difference in Ab titres wasat least partly due to smoking. To clarify the effects of smoking, we performed additional
analyses. However, the Brinkman index and the number of cigarettes per day did not
influence the Ab titres. Thus, smoking itself is a risk factor for low Ab titres, rather than the
duration of smoking or number of cigarettes per day. Moreover, smoking cessation can be
expected to effectively increase Ab titres because they were significantly lower in current
smokers than in ex-smokers. On the other hand, another study reported that alcohol was
the most impactful lifestyle factor, not smoking [10]. Although the authors of that study
do not show age-adjusted data, their conclusions were different from ours. Their results
were based on analysis of samples collected 2–5 weeks after the second dose of the vaccine.
In contrast, our samples were obtained 3 months after the second dose. One hypothesisis that alcohol may affect Ab production, whereas smoking may lead to decreases in thelevel of Abs after they are produced. In addition, we believe that an age-adjusted analysis
is critical. To clarify the reason for the difference between that study and ours, we need toanalyse longitudinal data to observe changes in Ab titres over time, and we are planningsuch an additional study. Although the mechanisms are not known, previous studies havereported an association between a smoking habit and lower Ab titres against both influenza
virus [23] and hepatitis B virus [24] following vaccination.
Some limitations and possible sources of bias in this study include the following:Firstly, the participants were limited in number and were all healthcare workers vaccinatedat a single national hospital in Tochigi prefecture, where the COVID-19 pandemic has
been well-controlled since SARS-CoV-2 began spreading around the globe. Therefore, theresults obtained in this study might not be generalisable on a wide scale, or even within
Japan. Secondly, several data, including body height and weight, were obtained by meansof a standardised structured self-reported questionnaire. We cannot deny the possibility
that some data may have been affected by recall bias. Third, the cut-off level of the Ab
against the SARS-CoV-2 spike antigen needs to be determined for the different variants of
SARS-CoV-2, but this information is currently unavailable. Further studies are needed toconfirm our observations.
In conclusion, we have reported, for the first time, real-world Ab titres against the
SARS-CoV-2 spike antigen at 3 months after the second dose of the BNT162b2 vaccine,which were much lower than those measured shortly after the second inoculation. We
demonstrated that the most important factors associated with low Ab titres were age andsmoking habit. In particular, current smoking status causes lower Ab titres, and smoking
cessation before vaccination may improve the individual effectiveness of the BNT162b2
vaccine. To establish a more personalised approach to vaccination involving earlier boosters,
different schedules or different types of vaccines, further studies are necessary regarding
the associations between Ab titres and the comprehensive medical histories of individuals.

Vaccines 2021, 9, 1042

8 of 9Author Contributions: Conceptualisation, 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., R.K. and M.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., R.K. and M.K.; visualisation, 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.
and T.T.; funding acquisition, T.T. and K.S. All authors have read and agreed to the published versionof 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, Minako Yamagishi, 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; SARS-CoV-2: severe acute respiratory syndrome
coronavirus 2; Ab: antibody; IQR: interquartile range.

References
1.
2.
3.
4.

5.

6.
7.
8.
9.

10.

11.
12.

Song, Z.; Xu, Y.; Bao, L.; Zhang, L.; Yu, P.; Qu, Y.; Zhu, H.; Zhao, W.; Han, Y.; Qin, C. From SARS to MERS, Thrusting Coronaviruses
into the Spotlight. Viruses 2019, 11, 59. [CrossRef]
Hoffmann, M.; Kleine-Weber, H.; Schroeder, S.; Krüger, N.; Herrler, T.; Erichsen, S. SARS-CoV-2 Cell Entry Depends on ACE2 andTMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 2020, 181, 271–280. [CrossRef] [PubMed]
Jiang, S.; Lu, L.; Liu, Q.; Xu, W.; Du, L. Receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets
for development of antiviral vaccines. Emerg. Microbes Infect. 2012, 1, e13. [CrossRef] [PubMed]
Dispinseri, S.; Secchi, M.; Pirillo, M.F.; Tolazzi, M.; Borghi, M.; Brigatti, C.; de Angelis, M.L.; Baratella, M.; Bazzigaluppi, E.;
Venturi, G.; et al. Neutralizing antibody responses to SARSCoV-2 in symptomatic COVID-19 is persistent and critical for survival.
Nat. Commun. 2021, 12, 2670. [CrossRef]
Lumley, S.F.; O’Donnell, D.; Stoesser, N.E.; Matthews, P.C.; Howarth, A.; Hatch, S.B.; Marsden, B.D.; Cox, S.; James, T.; Warren,
F.; et al. Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers. N. Engl. J. Med. 2021, 384, 533–540.
[CrossRef] [PubMed]
Miyakawa, K.; Jeremiah, S.S.; Kato, H.; Yamaoka, Y.; Go, H.; Yamanaka, T.; Ryo, A. Rapid detection of neutralizing antibodies to
SARS-CoV-2 variants in post-vaccination sera. medRxiv 2021. [CrossRef]
Plotkin, S.A. Correlates of protection induced by vaccination. Clin. Vaccine Immunol. 2010, 17, 1055–1065. [CrossRef]
Iyer, A.S.; Jones, F.K.; Nodoushani, A.; Kelly, M.; Becker, M.; Slater, D.; Mills, R.; Teng, E.; Kamruzzaman, M.; Garcia-Beltran, W.F.;
et al. Dynamics and significance of the antibody response to SARS-CoV-2 infection. medRxiv 2020. [CrossRef]
Chen, R.E.; Zhang, X.; Case, J.B.; Winkler, E.S.; Liu, Y.; VanBlargan, L.A.; Liu, J.; Errico, J.M.; Xie, X.; Suryadevara, N.; et al.
Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies. Nat. Med. 2021,
27, 717–726. [CrossRef] [PubMed]
Kageyama, T.; Ikeda, K.; Tanaka, S.; Taniguchi, T.; Igari, H.; Onouchi, Y.; Kaneda, A.; Matsushita, K.; Hanaoka, H.; Nakada, T.;et al. Antibody responses to BNT162b2 mRNA COVID-19 vaccine in 2015 healthcare workers in a single tertiary referral hospitalin Japan. medRxiv 2021. [CrossRef]
Müller, L.; Andrée, M.; Moskorz, W.; Drexler, I.; Walotka, L.; Grothmann, R.; Ptok, J.; Hillebrandt, J.; Ritchie, A.; Rabl, D.; et al.
Age-dependent immune response to the Biontech/Pfizer BNT162b2 COVID-19 vaccination. Clin. Infect. Dis. 2021. [CrossRef]
Terpos, E.; Trougakos, I.P.; Apostolakou, F.; Charitaki, I.; Sklirou, A.D.; Mavrianou, N.; Papanagnou, E.-D.; Liacos, C.-I.; Gumeni,S.; Rentziou, G.; et al. Age-dependent and gender-dependent antibody responses against SARS-CoV-2 in health workers andoctogenarians after vaccination with the BNT162b2 mRNA vaccine. Am. J. Hematol. 2021, 96, E257–E259. [CrossRef] [PubMed]Vaccines 2021, 9, 1042

13.

14.

15.
16.

17.
18.

19.

20.

21.

22.
23.
24.

9 of 9Kennedy, N.A.; Lin, S.; Goodhand, J.R.; Chanchlani, N.; Hamilton, B.; Bewshea, C.; Nice, R.; Chee, D.; Cummings, J.F.; Fraser, A.;et al. Infliximab is associated with attenuated immunogenicity to BNT162b2 and ChAdOx1 nCoV-19 SARS-CoV-2 vaccines inpatients with IBD. Gut 2021, 70, 1884–1893. [CrossRef]
Wei, J.; Stoesser, N.; Matthews, P.C.; Ayoubkhani, D.; Studley, R.; Bell, I.; Bell, J.I.; Newton, J.N.; Farrar, J.; Diamond, I.; et al.
COVID-19 Infection Survey team. Antibody responses to SARS-CoV-2 vaccines in 45,965 adults from the general population of
the United Kingdom. Nat. Microbiol. 2021, 6, 1140–1149. [CrossRef] [PubMed]
Ledford, H. How obesity could create problems for a COVID vaccine. Nature 2020, 586, 488–489. [CrossRef]
Watanabe, M.; Balena, A.; Tuccinardi, D.; Tozzi, R.; Risi, R.; Masi, D.; Caputi, A.; Rossetti, R.; Spoltore, M.E.; Filippi, V.; et al.
Central obesity, smoking habit, and hypertension are associated with lower antibody titres in response to COVID-19 mRNA
vaccine. Diabetes Metab. Res. Rev. 2021, e3465. [CrossRef]
Collier, D.A.; Ferreira, I.A.T.M.; Kotagiri, P.; Datir, R.P.; Lim, E.Y.; Touizer, E.; Meng, B.; Abdullahi, A.; Elmer, A.; Kingston, N.;et al. Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2. Nature 2021, 596. [CrossRef]
Monin-Aldama, L.; Laing, A.G.; Muñoz-Ruiz, M.; McKenzie, D.R.; del Molino del Barrio, I.; Alaguthurai, T.; Domingo-Vila, C.;
Hayday, T.S.; Graham, C.; Cooper, J.; et al. Interim results of the safety and immune-efficacy of 1 versus 2 doses of COVID-19
vaccine BNT162b2 for cancer patients in the context of the UK vaccine priority guidelines. medRxiv 2021. [CrossRef]
Perkmann, T.; Perkmann-Nagele, N.; Breyer, M.K.; Breyer-Kohansal, R.; Burghuber, O.C.; Hartl, S.; Aletaha, D.; Sieghart, D.;
Quehenberger, P.; Marculescu, R.; et al. Side-by-side comparison of three fully automated SARS-CoV-2 antibody assays with afocus on specificity. Clin. Chem. 2020, 66, 1405–1413. [CrossRef] [PubMed]
Callegaro, A.; Borleri, D.; Farina, C.; Napolitano, G.; Valenti, D.; Rizzi, M.; Maggiolo, F. Antibody response to SARS-CoV-2
vaccination is extremely vivacious in subjects with previous SARS-CoV-2 infection. J. Med. Virol. 2021, 93, 4612–4615. [CrossRef][PubMed]Pfizer. Pfizer and BioNTech Confirm High Efficacy and No Serious Safety Concerns Through Up to Six Months Following Second
Dose in Updated Topline Analysis of Landmark COVID-19 Vaccine Study. Available online: https://www.pfizer.com/news/
press-release/press-release-detail/pfizer-and-biontech-confirm-high-efficacy-and-no-serious (accessed on 15 June 2021).
Lopez Bernal, J.; Andrews, N.; Gower, C.; Gallagher, E.; Simmons, R.; Thelwall, S.; Stowe, J.; Tessier, E.; Groves, N.; Dabrera, G.;
et al. Effectiveness of Covid-19 Vaccines against the B.1.617.2 (Delta) Variant. N. Engl. J. Med. 2021, 385. [CrossRef] [PubMed]
MacKenzie, J.S.; MacKenzie, I.H.; Holt, P.G. The effect of cigarette smoking on susceptibility to epidemic influenza and on
serological responses to live attenuated and killed subunit influenza vaccines. J. Hyg. 1976, 77, 409–417. [CrossRef] [PubMed]
Zimmermann, P.; Curtis, N. Factors that influence the immune response to vaccination. Clin. Microbiol. Rev. 2019, 32, e00084-18.
[CrossRef] [PubMed]