Article

Kinetics of Anti-SARS-CoV-2 Antibody Response Following
Two Doses of the BNT162b2 mRNA Vaccine: A JapaneseSingle-Center Primary Care Clinic Report Involving Volunteers
and Patients with Autoimmune Disease
Tomoko Sugiura 1,2, *, Hiroaki Sugiura 2 , Hiroaki Kato 1 , Yuko Nariai 1 , Yuuki Mizumoto 1 , Kozue Hanada 2 ,Rieko Takahashi 2 , Yukari Hinotubo 2 , Naoko Tanaka 2 , Mutsumi Sasaki 2 , Haruki Eguchi 3 , Hiroki Kamino 1,4
and Takeshi Urano 1,4,5
1

2
3
4
5

*

Citation: Sugiura, T.; Sugiura, H.;
Kato, H.; Nariai, Y.; Mizumoto, Y.;
Hanada, K.; Takahashi, R.; Hinotubo,
Y.; Tanaka, N.; Sasaki, M.; et al.
Kinetics of Anti-SARS-CoV-2
Antibody Response Following Two
Doses of the BNT162b2 mRNA
Vaccine: A Japanese Single-Center
Primary Care Clinic Report Involving
Volunteers and Patients with
Autoimmune Disease. Infect. Dis. Rep.
2023, 15, 24–33. https://doi.org/
10.3390/idr15010003

Department of Biochemistry, Faculty of Medicine, Shimane University, 89-1, Enya-cho,
Izumo City 693-0021, Japan
Sugiura Clinic, 2-8-3, Kitahon-machi, Imachi-cho, Izumo City 693-0002, Japan
Eguchi Clinic, 6-43, Enya-Arihara-cho, Izumo City 693-0023, Japan
mAbProtein Co., Ltd. 89-1, Enya-cho, Izumo City 93-0021, Japan
Vaccines and Therapeutic Antibodies for Emerging Infectious Diseases, Shimane University,
Izumo City 693-0021, Japan
Correspondence: sugiura-q@hi.enjoy.ne.jp; Tel.: +81-853-20-2127

Abstract: Despite the promising effectiveness of the coronavirus disease 2019 vaccination using anmRNA vaccine, the short efficacy duration and some poor responses to the vaccination remain major
concerns. We aimed to clarify the monthly kinetics of the anti-SARS-CoV-2 spike receptor-binding
domain antibody response after two doses of the BNT162b2 vaccine in a Japanese population. A
chemiluminescent enzyme immunoassay (CLIA) and an enzyme-linked immunosorbent assay wereused to measure the antibody levels in 81 Japanese adults (age, <65 years). The antibody levels
increased 10-fold at 2–3 weeks following the second dose of BNT162b2 and declined thereafter toapproximately 50%, 20%, and 10% of the peak levels at 2, 3, and 6 months, respectively. To compare
the antibody titers among different groups, older adults (age, >65 years; n = 38) and patients withsystemic lupus erythematosus (SLE, n = 14) were also investigated. A decline in the mean relative
antibody titers was observed in older men compared with younger men and in patients with SLE
compared with individuals aged <65 years. Although the antibody levels increased drasticallyfollowing two BNT162b2 doses, they then declined rapidly. Furthermore, poor responders to thevaccination were observed. Repeated vaccinations are required to maintain high antibody levels.
Keywords: BNT162b2; COVID-19 vaccination; Japanese people; older men; systemic lupus
erythematosus

Academic Editor: Nicola Petrosillo
Received: 22 October 2022
Revised: 7 December 2022
Accepted: 18 December 2022
Published: 26 December 2022

Copyright: © 2022 by the authors.
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/).

1. Introduction
mRNA vaccines have been implemented worldwide following the outbreak of thecoronavirus disease (COVID-19) pandemic. A two-dose regimen of the BNT162b2 vaccine by Pfizer-BioNTech confers 95% protection against COVID-19 [1]. In Japan, public
vaccination began in February 2021, and according to the Ministry of Health, Labour, andWelfare of Japan, 79% of the total population (100 million people) had received two vaccine
doses by February 2022. Furthermore, 50% of the total population received a third dosein May 2022. In addition to group vaccinations in public spaces, primary-care physicians
administered individual vaccinations in Japan.
One major issue following the mRNA vaccination for COVID-19 concerns the vaccine’s
ability to confer long-term immunity. Several reports have provided evidence that indicatesthe antibody titers increase dramatically following the administration of two doses of the

Infect. Dis. Rep. 2023, 15, 24–33. https://doi.org/10.3390/idr15010003

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

Infect. Dis. Rep. 2023, 15

25mRNA vaccine; however, these titers then decrease rapidly [2–5]. As the antibody titers
decrease, so does the clinical effectiveness of the vaccination [6]. Thus, repeated booster
vaccinations within a short interval are crucial to prevent disease and community spread.
Another issue with regard to the COVID-19 vaccination concerns interindividual
differences in vaccine immunity. Even in a healthy population, the range of antibody
titers after vaccination is widely distributed. Some factors, such as male sex or alcohol
consumption habits, may influence interindividual differences [7]. In certain groups, suchas older adults and those with autoimmune diseases or cancer, some individuals havereportedly shown an inadequate immune response to the COVID-19 vaccination and werepoor responders [7–13].
Detection systems for anti-SARS-CoV-2 spike receptor-binding domain (RBD) antibodies are readily available in the Japanese healthcare system. The chemiluminescent enzyme
immunoassay (CLIA) produced by Abbott Laboratories is one such system used worldwide.
Nevertheless, data on the normal range of the antibody titers for the SARS-CoV-2 spike
RBD after two doses of the COVID-19 vaccination in healthy Japanese individuals arelimited. A high antibody titer has been shown to correlate with solid immunity against
COVID-19 [6], although antibody production reflects only a part of the vaccine immunity.
This study aimed to assess the monthly mean antibody titer in Japanese adults aged
<65 years after the second dose of BNT162b2. Through converting antibody titers into amean titer percentage in relation to Japanese individuals aged <65 years, we compared
the antibody titers between different groups, such as older adults or patients with anautoimmune disease, regardless of the time of the titer determination after the second dose.
2. Materials and Methods
2.1. Study Design
This single-center prospective study, conducted in Izumo City, Shimane Prefecture,
Japan, involved individuals and patients who had received SARS-CoV2-mRNA vaccinations with Pfizer-BioNTech BNT162b2 (first and second doses) from April to October 2021
at medical clinics and public spaces. During this period, approximately 3000 vaccinations
were administered to 1500 outpatients and healthy individuals residing in the vicinity of
the Sugiura Clinic.
Blood samples for antibody testing were collected four times: on the day of the second
vaccination (one dose), 2–3 weeks after the second dose (peak), 3 months after the second
dose, and 6 months after the second dose of vaccination. Individuals who underwent atleast two blood collections at a different time, other than the four previously mentioned
time points, were included in the study. Information on all of the participants was collected,
including medical information, smoking and alcohol consumption habits, and side-effectsof vaccination, such as fever and total IgG serum levels. The study was approved by the
Ethics Committee of MEDIKS (Sapporo, Japan) with approval no. MX-4150FN-151448 and
the Shimane University Institutional Committee of Ethics (Shimane, Japan) with approval
no. 4913. Written informed consent was obtained from all participants.
2.2. Participants
Inclusion criteria comprised volunteers aged 20–65 years who had received a BNT162b2
vaccination at the Sugiura Clinic from 14 September to 31 October 2021 and who provided
written informed consent to participate in this study. Those with a history of COVID-19 or
those who had received immunosuppressive therapies were excluded. In total, 81 healthy
individuals aged <65 years (39 men, 42 women) were enrolled. The mean age ± standard
deviation (SD) was 42.2 ± 11.9 years. We also included 38 individuals (age, >65 years; men,50%) who attended the Sugiura Clinic with chronic diseases, such as hypertension but
excluded those with autoimmune diseases. The mean age ± SD of the older adult groupwas 78.2 ± 6.0 years, and this group had not received immunosuppressive therapy. Furthermore, 14 patients with systemic lupus erythematosus (SLE) (women, 100%) who attended
the Sugiura Clinic and received immunosuppressive treatments were also recruited. All ofInfect. Dis. Rep. 2023, 15

26

the patients with SLE were in remission, and the mean SLE disease activity index (SLEDAI)
was 1.14 ± 1.45. The mean age ± SD in this group was 42.2 ± 11.9 years, and all of thepatients had received a median dose of 5.6 ± 3.0 (range, 1–12) mg/day of oral prednisone.
Other standard immunosuppressive treatments included hydroxychloroquine in 11/14(78.5%) patients and tacrolimus in 10/14 (71.4%) patients. All of the participants, including the volunteers, older adults, and patients with SLE, provided their written informed
consent prior to participation in this study.
2.3. Measurement of Antibody Titers
The anti-SARS-CoV-2 spike RBD antibody titers were assessed using two detection
systems. The CLIA, from Abbott Laboratories (Chicago, IL, USA), is used worldwide andwas obtained under contract by Nihon Rinsho, Inc. (Kyoto, Japan) for commercial antibody
testing. This system detects the anti-SARS-CoV-2 spike RBD IgG using an ARCHITECT
i2000R analyzer (Abbott Laboratories). The cutoff and upper detection limits were 50
and 80,000 arbitrary units (AUs)/mL, respectively. The second detection system was asandwich enzyme-linked immunosorbent assay (ELISA) system (E-S-001), newly developed
by mAbProtein, Inc. (Izumo City, Shimane, Japan) to detect anti-SARS-CoV-2 spike RBD
IgG type antibodies in the serum. The lower detection limit was 0.4 AU/mL, which was
determined by adding two standard deviations to the mean optical density (OD) obtained
from 50 assays of the zero standard. All the samples in the study were measured using
the CLIA (Abbott) and ELISA (mAbProtein) systems, with the antibody titers showing asignificant correlation (r = 0.841, p < 0.001, Pearson’s test).
2.4. Kinetic Graph
A program using the R language, https://CRAN.R-project.org/package=AntibodyTiters (accessed on 30 September 2021), enabled us to visualize the raw antibody data at
any sampling point in the form of kinetic graphs. At least two blood sampling points are
required to generate a kinetic graph. The kinetic charts showed increases and decreases inantibody titers for individuals generally or for different categories of individuals, such as
those aged <65 years or ≥65 years.
2.5. Antibody Data Comparison
Following data collection, the monthly mean titers following two doses of BNT162b2
in Japanese individuals aged <65 years were determined. The raw data of antibody titersof older individuals and patients with SLE were changed to percentages relative to themean titer of the same sampling time as the individuals aged <65 years. At least two
blood samples were obtained per person, and the mean percentage was calculated using
two or more raw antibody titers. To compare healthy individuals and different categories
of individuals, the mean percentages of the relevant groups were assessed. Multiple
regression analysis was performed to determine the factors influencing high and low
antibody level percentages.
2.6. Statistical Analysis
The data for the groups were compared using a Student’s t-test after confirming theraw data were normally distributed, whereas the inter-group data were compared using
either Pearson’s or Spearman’s tests. All tests were two-sided, and a p-value < 0.05 wasconsidered statistically significant. Statistical analyses were performed using GraphPad
Prism, Version 9.3.1 (GraphPad Software, San Diego, CA, USA) software.
3. Results
3.1. Kinetics of Antibody Response
Figure 1 shows the changes in the antibody titers over time in volunteers and individuals aged <65 years. The data are presented as the geometric mean at each time of theblood collection. Although the baseline data at pre-vaccination are unavailable, individuals

3. Results
3.1. Kinetics of Antibody Response
Infect. Dis. Rep. 2023, 15

Figure 1 shows the changes in the antibody titers over time in volunteers andindi27viduals aged <65 years. The data are presented as the geometric mean at each time of theblood collection. Although the baseline data at pre-vaccination are unavailable, individualswho
whohadhadnotnotreceived
received
the
COVID-19
vaccinationor whonever
infectedthe
COVID-19
vaccinationor whohad hadneverbeenbeen
infected
with with
COVID-19
showednodetectableantibodies
tothe
SARS-CoV-2
RBD;
this
was
confirmed
COVID-19 showed no detectable antibodies to the SARS-CoV-2 RBD; this was confirmed
throughexaminingfrombefore
beforethe
theCOVID-19
COVID-19
pandemic(data
not shown).
throughexamining50
50pooled
pooled sera
sera frompandemic(data
not shown).

FigureKinetics of
spikespike
RBD antibody
response after
two dosesof theFigure1.1.Kinetics
ofanti-SARS-CoV-2
anti-SARS-CoV-2
RBD antibody
response
after
twoBNT162b2
doses of the
BNT162b2
titers
at of
each
time
of blood
presented
as mean,the geometricvaccine. vaccine.
AntibodyAntibody
titers at each
time
blood
collection
arecollection
presentedareas thegeometricasmean,
as measured(A) (B)
CLIA
andsystems(B) ELISA
systemsaged
in individuals
aged time<65 years.
measuredusing (A)using
CLIA and
ELISA
in individuals<65 years. X-axis,of bloodX-axis,
timeof bloodY-axis,
collection;
Y-axis, anti-SARS-CoV-2
spiketiter
RBD
S antibody(AU/mL);AU, arbicollection;
anti-SARS-CoV-2
spike RBD S antibody(AU/mL);AU, titer
arbitraryunit; CLIA,
trary
unit; CLIA, chemiluminescent
enzymeimmunoassay;
ELISA,
enzyme-linked
immunosorbent
chemiluminescent
enzyme immunoassay;
ELISA,
enzyme-linked
immunosorbent
assay; M,months.
assay; M, months.
The first blood samples were collected from individuals aged <65 years on the dayof Thereceiving
the second
dosewere(3 weeks
after fromthe first
dose) and aged
expressed
as “onefirst blood
samplescollected
individuals<65 yearson dose”
the day of(Figure 1). The mean ± SD and median (a 95% confidence interval [CI]) antibody titers
receiving the second dose (3 weeks after the first dose) and expressed as “one dose” (Figwere 1253 ± 1098 IU/mL and 917 (720–1202) IU/mL, respectively, using the CLIA sysuretem,1). Themean ± SD and median (a 95% confidence interval [CI]) antibody titers wereand 96.4 ± 89.4 IU/mL and 77.2 (95% CI, 720–1202) IU/mL, respectively, using the
1253
±
1098
IU/mL
and
917 (720–1202)
IU/mL,
respectively,
using2–3
theweeks
CLIAafter
system,ELISA system.
The
antibody
titers showeda significant
elevation
the and96.4± 89.4dose,
IU/mL
and
77.2 and(95%ELISA-derived
CI, 720–1202)means
IU/mL,
using
secondwith
CLIA± respectively,
SD and median(95%theCI)ELISA
titers system.
antibody
titers showeda significant
elevation
the secondof The12,406± 8014 IU/mL
and 10,983(7171–13,045)
IU/mL;2–3
andweeks
1356.5after± 1461.2
IU/mLdose,
andCLIA759.1 (512.3–931.7)
IU/mL, respectively.
peak titers
werehigher± 8014
withand ELISA-derived
means ± SDTheseand median(95%CI)significantly
titers of 12,406(approximately
times) than thoseat timepoint
0 (a ±p 1461.2< 0.001 IU/mL
with theandStudent’s
for
IU/mL
and 10,98310(7171–13,045)
IU/mL;
and1356.5759.1 t-test(512.3–931.7)
bothmethods
ofdetection).
However,theantibody
titersmeasured
using
the
CLIA
systemIU/mL, respectively. These peak titers were significantly higher (approximately 10 times)
decreased rapidly to approximately 50% and 20% of the peak titers at 2 and 3 months,
than those at time point 0 (a p < 0.001 with the Student’s t-test for both methods of detecrespectively, after the second dose. Subsequently, the antibody titers slowly dropped betion).
However, the antibody titers measured using the CLIA system decreased rapidly to
low 1/10 of the peak titers 6 months after the second dose. The monthly antibody titers
approximately
50%
20%
ofELISAthe peaktiters are
at 2presented
and 3 months,
after themeasured using
theandCLIA
andmethods
in Tablerespectively,
1. As previously
seconddose.theSubsequently,
antibody
titerswere
slowlydroppedbelow
1/10
ofpeak,the peakdescribed,
blood samples thefor antibody
testingcollected
four times:
one
dose,
titers6monthsafter
theseconddose.
Themonthly
antibody
titersmeasuredusing
and at 3 and 6 months after the second dose. Those who underwent at least two blood thecollectionsat anymethods
time pointare
wereincluded inin thestudy.
Therefore,
the monthly
antibody
CLIA
and ELISA
presented
Table
1. As
previously
described,
the bloodtiters were
determinedtesting(Table 1).
samples
for antibody
were collected four times: one dose, peak, and at 3 and 6

months after the second dose. Those who underwent at least two blood collections at any
3.2. Interindividual Differences in Antibody Titers

In this study, the Japanese individuals aged <65 years showed a considerable variationin their antibody titers. The peak antibody titers at 2–3 weeks after the second dose ranged
from 3362 IU/mL to 34,386 IU/mL using the CLIA system, with an approximately 10-folddifference between the individual titers. Similarly, the peak antibody titers ranged from150.1 IU/mL to 7160 IU/mL using the ELISA system, with an approximately 45-folddifference between the individual titers. Notably, individual differences in the antibody
titers were maintained throughout the study period. Those with relatively high peak
antibody titers tended to maintain higher titers at subsequent time points. A significant

Infect. Dis. Rep. 2023, 15

28

correlation was noted between an individual’s antibody titers at time 0 and the peak, at thepeak and 3 months after vaccination, and at 3 and 6 months after vaccination (r = 0.768 by
the Pearson’s correlation test and a p < 0.001 for all the comparisons). Multiple regression
analysis showed that a fever >37.5 ◦ C, a major side-effect of the vaccination, occurred in 21
of 81 individuals (25.9%) and was related to higher antibody titers in individuals aged <65
years (p = 0.03) but was not otherwise related to age, smoking, alcohol consumption, sex,
and serum IgG levels.
Table 1. Monthly mean and median titers of the anti-SARS-CoV-2 spike RBD antibody in individuals
aged <65 years after receiving two doses of BNT162b2.
Timing of Blood Sampling
after the Second Dosen

Abbott (CLIA)
Mean ± SD
Median (95% CI)(AU/mL)(AU/mL)The same day (time point 0)
2–3 W after (peak point)
2 M after (42–67 days)
3 M after (68–97 days)
4 M after (98–125 days)
5 M after (126–153 days)
6 M after (154–181 days)

50
53
18
56
18
16
51

1253 ± 1098
12,406 ± 8014
6030 ± 4332
2322 ± 1751
2305 ± 1204
1296 ± 656
945 ± 662

917 (720–1202)
10,983 (7171–13,045)
5302 (2941–7337)
2129 (1638–2409)
2304 (1208–3061)
1083 (768–1734)
888 (585–990)

mAbProtein (ELISA)
Mean ± SD
Median (95% CI)(AU/mL)(AU/mL)96.4 ± 89.4
1356.5 ± 1461.2
363.8 ± 226.1
163.9 ± 121
98.2 ± 59.7
83.7 ± 50.7
48.7 ± 34.6

77.2 (61.1–101.6)
759.1 (512.3–931.7)
366.9 (201–524.3)
145.4 (117–172)
91.3 (60.4–153)
81.5 (59–133.3)
42.7 (32.5–55)

AU, arbitrary unit; CI, confidence interval; CLIA, chemiluminescent enzyme immunoassay; ELISA, enzyme-linked
immunosorbent assay; M, months; SD, standard deviation; W, weeks.

3.3. Antibody Titers in the Older Population

Infect. Dis. Rep. 2022, 14,

Using the R program, we plotted the kinetics of the antibody response in the older
adult population aged >65 years and compared them with that of healthy individuals aged
<65 years (Figure 2). At the commencement of the project, the older adult population hadcompleted two vaccination doses; therefore, there was no baseline data concerning theirpeak titers. The older adult individuals had relatively lower antibody titers than those aged
<65 years nearly 3 months after the second vaccination.

6

FigureKineticsof ofanti-SARS-CoV-2
spike
RBDresponse
antibody
responseolder
adults. The
Figure 2. Kinetics
the theanti-SARS-CoV-2
spike RBD
antibody
in olderadults.inThegeometric
geometric
mean in individuals
aged>65and(brownlines)
andyears<65 after(bluetwolines)
years
two doses ofmean in individuals
aged >65 (brownlines)<65 (bluelines)
dosesof theafter
BNT162b2
the
BNT162b2
vaccinemeasured
using
the
CLIA
(B)inELISA
is shown
figure. Data for
vaccinemeasuredusing
the (A) CLIAand(B)(A)ELISA
is andshown
this figure.
Datainforthisindividuals
individuals
agedwere>65 obtained
years wereobtainedmonthstheythehadreceivedseconddose. AU,
aged >65 years
3 months
after3 they
had after
receivedseconddose.theAU,
arbitrary
arbitrary
CLIA, chemiluminescent
enzymeimmunoassay;immunosorbent
ELISA, enzyme-linked
unit; CLIA,unit;
chemiluminescent
enzyme immunoassay;
ELISA, enzyme-linked
assay;
immunosorbent
assay; M, months.
M, months.

To compare the antibody titers of the older individuals with those of the young
individuals, we changed all the raw antibody titers into percentages of the monthly mean
titer (Table 1) of people aged <65 years, as previously described. The mean percentage ofthe antibody titers in the group aged >65 years (n = 38) was 84.6 ± 118.9%, which wasInfect. Dis. Rep. 2023, 15

Infect. Dis. Rep. 2022, 14,29

To compare the antibody titers of the older individuals with those of the young
individuals, we changed all the raw antibody titers into percentages of the monthly mean
titer (Table 1) of people aged <65 years, as previously described. The mean percentageof the antibody titers in the group aged >65 years (n = 38) was 84.6 ± 118.9%, which
was comparable to that of the group aged <65 years (98.9 ± 67.4%) (Figure 3). However,when stratified according to sex, the mean titers were significantly lower in the men
aged >65 years than in those aged <65 years (44 ± 59.8% vs. 94 ± 72.9%, difference:−49.9 ± 14.4%, 95% CI −78.5–−23.3, p = 0.0007, Student’s t-test). Conversely, no significant
7
differences were observed between the mean antibody titers in the women aged <65 years(104 ± 60.6%) and those aged >65 years (120 ± 145.3%) (Figure 3).

Figure 3. The anti-SARS-CoV-2 spike RBD antibody titers in younger vs. older individuals. Each
Figure 3. The anti-SARS-CoV-2 spike RBD antibody titers in younger vs. older individuals. Eachantibody titer measured using the CLIA (Abbott) system was expressed as a percentage of theantibody titer measured using the CLIA (Abbott) system was expressed as a percentage of themonthlymean
meanantibody
antibodytiter
titerininindividuals
individualsaged
aged<65<65years
years(as(asshown
shownininTable
Table1).
1).(A)(A)TheThemeanmean
monthly
percent
antibody
titersintheindividuals
aged
<65
years
(98.9
±
67.4%)
and
those
aged
>65
years
percent antibody titers in the individuals aged <65 years (98.9 ± 67.4%) and those aged >65 years(84.6±±
118.9%)
arecomparable,comparable,withwithnonosignificant
significantdifference.
difference.(B)(B)When
Whenstratified
stratifiedaccording
accordingtotosex,
sex,
(84.6
118.9%)
arethetheantibody
antibodytiters
titerswere
weresignificantly
significantlylower
lowerininmen
menagedaged >65>65years
years(44(44±±59.8%)
59.8%)compared
comparedwith
withmenmen
aged
72.9%)(Student’s
t-test,p =p 0.0007;
difference
in in
means
= −49.9± 14.4%;
95%95%
CI
aged<65<65years
years(94(94± ±
72.9%)(Student’s
t-test,= 0.0007;differencemeans= −49.9± 14.4%;−78.5–−23.3).
(C) Contrastingly,
there isno significant
differencein the mean
antibody
titersCI −78.5–−23.3).
(C) Contrastingly,
there
is no significant
differencein the percent
mean percent
antibody
betweenwomenwomen
aged <65years± 60.6%)
women>65 aged
years>65(120years± 145.3%).
nottiters between
aged<65(104
years(104 ±and60.6%)
andagedwomen(120 ±N.S.,
145.3%).
significant; CI, confidence interval; CLIA, chemiluminescent enzyme immunoassay; RBD, receptorN.S., not significant; CI, confidence interval; CLIA, chemiluminescent enzyme immunoassay; RBD,
binding domain.
receptor-binding domain.

3.4. Immunosuppressed Patients
Fourteen patients with SLE who visited the Sugiura Clinic for immunosuppressive
treatments were included in this study. All 14 patients were women aged <65 years (mean± SD years, 42.2 ± 11.9) who had received a median dose of 5.6 ± 3.0 (range, 1–12) mg/dayof oral prednisone. Other standard immunosuppressive treatments includedhydroxychloroquine in 11/14 (78.5%) patients and tacrolimus in 10/14 (71.4%) patients.

Infect. Dis. Rep. 2023, 1530

3.4. Immunosuppressed Patients

Infect. Dis. Rep. 2022, 14,

Fourteen patients with SLE who visited the Sugiura Clinic for immunosuppressive
treatments were included in this study. All 14 patients were women aged <65 years(mean ± SD years, 42.2 ± 11.9) who had received a median dose of 5.6 ± 3.0 (range,
1–12) mg/day of oral prednisone. Other standard immunosuppressive treatments includedhydroxychloroquine in 11/14 (78.5%) patients and tacrolimus in 10/14 (71.4%) patients. The
blood collection times following the two vaccine doses were not the same for all patients;
therefore, we expressed the antibody titers in the patients with SLE as a percentage of those8
in healthy individuals aged <65 years at the same time point. At least two blood samples
were obtained per patient, and the mean of the percentages relative to individuals <65
years was calculated.
The
Themeanmeanpercentage
percentageofofanti-SARS-CoV-2
anti-SARS-CoV-2spike
spikeRBD
RBDantibody
antibodytiters
titersininpatientspatientswith
withSLE± 71.0%,whichwaswassignificantly
significantly
lower
than
in individuals<65
SLEwaswas 58.2
58.2 ±
71.0%, which
lower
than
thatthatin individuals
agedaged<65 years
years± 67%)(Student’s
p = 0.0037;
the difference
betweenthe means,−40.7±(98.8 (98.8± 67%)(Student’s
t-test,t-test,
p = 0.0037;
the difference
betweenthe means,−40.7 ±
13.8%,
13.8%,
95%
CI −68–−13)(Figure4A).
Notably,somepatientswith
withSLESLE showed
showed only aaslight
95% CI−68–−13) (Figure
4A).
Notably,somepatients
slightincrease
theantibody
antibodytiters
titersafter
the second
below
increase in theafter
the second
dose, dose,
whichwhich
rapidlyrapidly
declineddeclined
below detection
detectionlevelswithin6months.
The
antibody
titersin
5/14(35.7%)
patientswith
SLEoflevels within 6 months. The antibody titers in 5/14 (35.7%) patients with SLE were <10%
wereof those inaged
individuals
aged <65thethroughout
the study period.
those<10%in individuals<65 throughout
study period.

Figure 4. (A) The mean percent of antibody titers in patients with SLE (58.2 ± 71.0%) is significantly
Figure 4. (A) The mean percent of antibody titers in patients with SLE (58.2 ± 71.0%) is significantly
lowerthan
thanthatthatininindividuals
individualsaged
aged<65<65years
years(98.8(98.8±±67%)67%)(Student’s(Student’st-test,
t-test,pp==0.0037;
0.0037;difference
difference
lower
mean
titers:−40.7±
13.8%;
95%
CI
−
68–
−
13).
(B)
A
significant
correlation
betweenthepercent
mean titers: −40.7 ± 13.8%; 95% CI −68–−13). (B) A significant correlation between the percent
antibody
titers
and
total
IgG
levelsinpatients
with
SLE
is
seen
(r
=
0.75,
95%
CI
0.51–0.88,
p<
0.0001).
antibody titers and total IgG levels in patients with SLE is seen (r = 0.75, 95% CI 0.51–0.88, p < 0.0001).
CI,confidence
confidenceinterval;interval;RBD,
RBD,receptor-binding
receptor-bindingdomain.
domain.
CI,

The correlation
correlation between
between thetitersandandthe thedosedoseof oralThe
the percentage
percentageofofantibody
antibody
titersof predoralnisoneinthesepatients
wasofborderline
significance(r=−
0.577,
p=0.031,
Spearman’s
prednisone in these patients was of borderline significance (r = −0.577, p = 0.031,
correlation correlationtest). Furthermore,
we assessed
the totalthe
serumlevels(normalrange,
Spearman’stest). Furthermore,
we assessed
total IgG
serum
IgG levels(normal
800–1780
mg/mL)
inpatients
with
SLE,
which
are
reflectiveof
their
immunosuppressive
range, 800–1780 mg/mL) in patients with SLE, which are reflective of theirstate. The mean total
IgG
in meanpatients
was 1168241wasmg/dL,
that
immunosuppressive
state.
The
totalwithIgG SLE
in patientswith±SLE
1168 ±whereas
241 mg/dL,
inindividuals
aged
<65
years
was
1241
±
207
mg/dL;
however,this
differencewasnotwhereas that in individuals aged <65 years was 1241 ± 207 mg/dL; however, this difference
statisticallysignificant.
In
2/14
patients
with
SLE,
the
serum
IgG
levels
were
lower
than
the
was not statistically significant. In 2/14 patients with SLE, the serum IgG levels were lower
normallimit,
whereasthey
were
withinanormalrange
inhealthy
individuals.
Astrongthan the normal limit, whereas they were within a normal range in healthy individuals. A
correlation
was foundbetweenthe anti-SARS-CoV-2
spikespikeRBDRBD
antibody
titerstiters
and andtotal
strongcorrelation
was foundbetweenthe anti-SARS-CoV-2
antibody
IgG
levels
(r
=
0.75,
p
<
0.0001,
Spearman’s
correlation
test;
Figure4B)
inpatients
withSLE
total IgG levels (r = 0.75, p < 0.0001, Spearman’s correlation test; Figure 4B) in patients with
but
not
in
individuals
aged<65
years.
We
performed
multipleregression
tests
todetermine
SLE but not in individuals aged <65 years. We performed multiple regression tests tothe factors associated with impaired antibody production after vaccination in patients withdetermine the factors associated with impaired antibody production after vaccination in
SLE. Only the serum total IgG levels showed an association (t = 2.76, p = 0.0157), whereas
patients with SLE. Only the serum total IgG levels showed an association (t = 2.76, p =the prednisone dose, age, or other immunosuppressive agents showed no association.
0.0157), whereas the prednisone dose, age, or other immunosuppressive agents showedno association.

4. Discussion
Our study design was similar to that of Naaber et al., who assessed the levels of anti-

Infect. Dis. Rep. 2023, 15

31

4. Discussion
Our study design was similar to that of Naaber et al., who assessed the levels ofanti-SARS-CoV-2 spike RBD antibodies in 122 Estonian healthcare workers using the CLIA
system [4]. Consistent with their findings, we found a significant increase in antibody titers(10-fold in the present study vs. 19-fold in the study by Naaber et al.) after the second doseof BNT162b2, as well as a subsequent rapid decrease to 50% of the peak titer at 2 months
and 20% at 3 months (in both studies). Subsequently, the antibody titers declined gradually
to 1/10 (the present study) or 1/20 (Naaber et al.) of the peak titers 6 months after thesecond dose. The rapid decay of the antibody titers for up to 3 months and a gradualdecline during the following 3 months have repeatedly been reported in other studies [2–6].
This pattern might reflect the initial humoral B and T-cell immune response generated
through the two doses of the vaccine [14], the half-life of the human immunoglobulin [15],and the existence of memory B cells, which continuously produce small amounts of specificimmunoglobulin for the SARS-CoV-2 spike RBD, as is the case following a COVID-19
infection [16,17].
Researchers have concentrated on investigating the duration of the neutralization
activity shown through vaccination-induced antibodies. According to Naaber et al., theinhibition of the S protein of five variants of concern (wild-type, Alpha, Beta, Gamma,
Delta, and Kappa) and the binding of the angiotensin-converting enzyme 2 (ACE2) receptor
in an in vitro ELISA waned at 3 months after the second dose [4]. Another study involving
225 healthy Japanese medical workers who received two doses of BNT162b2 found that thehalf-life for neutralizing the activity to block SARS-CoV-2 virions from infecting target cells
determined using immune sera was 67.8 days [3]. A test-negative study conducted from
May to September 2021 evaluated the duration of protection from COVID-19 conferredthrough two doses of BNT162b2 in a large Israeli population [6]. During that study period,
the Delta variant was prevalent in Israel, and an increased odds ratio of COVID-19 RT-PCR
positivity was observed in individuals 146 days after BNT162b2 vaccination compared withthat noted in individuals <146 days after BNT162b2 vaccination [6]. Based on these data
concerning the Delta variant, a two-dose BNT162b2 vaccination appears to be effective forapproximately 3–4 months.
The serum levels of the anti-SARS-CoV-2 spike RBD antibody and its neutralizing
activity after vaccination reflect the different aspects of acquired humoral immunity; however, several studies have shown a close association between the two [3,4]. A high antibody
titer equates to a high neutralizing activity. In our study, the mean antibody titer 3 months
after the second dose was 2332 ± 1751 IU/mL with the CLIA and 163.9 ± 121 IU/mL with
the ELISA. These values might indicate the minimum titer required for protection fromCOVID-19, particularly the Delta variant.
In this study, older adult men showed a poor response to the COVID-19 mRNA
vaccination. Aging is a significant risk factor for severe COVID-19 [18]. Although several
studies have reported an impaired immune response to the COVID-19 vaccine in older
individuals [7,13], older COVID-19-recovered individuals show improved antibody andcellular responses, similar to younger individuals [13]. Therefore, booster doses are likely tobe effective in the older population. Modifying the vaccination protocol, such as shortening
the interval between doses or increasing the mRNA dose, might help improve the immune
response in high-risk groups.
We selected SLE as a disease model. In a study involving 90 patients with SLE who
had received two doses of BNT162b2, Izmirly et al. reported that 29% of the patients
produced significantly lower levels of anti-SARS-CoV-2 spike RBD antibodies comparedwith the healthy controls [11]. Other investigators have reported that 13.2% of patientswith autoimmune systemic diseases were non-responders to the COVID-19 vaccination [9].
In autoimmune inflammatory rheumatic diseases, immunosuppressive therapies, such as
glucocorticoids, rituximab, mycophenolate mofetil, and abatacept, can impede the detectionof anti-SARS-CoV-2-specific antibodies [10,19]. In the present study, 5/14 (35.7%) patients
showed extremely low titers of anti-SARS-CoV-2 spike RBD antibodies, namely, <10% ofInfect. Dis. Rep. 2023, 15

32

those in healthy individuals aged <65 years. Overall, the mean percentage of the antibody
titers was <50% in patients with SLE than in healthy Japanese individuals aged <65 years.
Initially, we focused on the association of the antibody titers with the prednisone dosage
in these patients; however, a multiple regression model found that they were significantly
associated with the total IgG levels. Low total IgG levels might be associated with impaired
B cell function, primarily due to long-term oral prednisone [19], even at reduced doses.
The main limitation of our study was its small sample size. When we initiated thestudy, the two-dose vaccination regimen had already started for the Japanese population.
Nonetheless, we obtained at least 50 samples for the time 0, the peak, 3 months, and 6
months after the second vaccination. Due to significant interindividual differences, even in
the healthy younger group, the mean and median titers of the antibodies always showed aconsiderable difference.
5. Conclusions
Some concerns associated with COVID-19 vaccination using the mRNA vaccine
BNT162b2 have been raised. This study found, first, that there was a rapid decline in
the antibody titers after the second dose, requiring additional vaccinations at brief intervals.
Second, some groups, such as older adult men or patients receiving immunosuppressive
therapy, might be poor responders to vaccination. Finally, as booster vaccination has beenreported to be effective in maintaining high antibody levels, modified protocols, such as
applying shorter intervals or increasing the vaccine dosage for high-risk groups, might
be required.
Author Contributions: Conceptualization, T.S. and T.U.; methodology, T.S.; software, H.K. (HiroakiKato) and Y.M.; validation, H.K. (Hiroki Kamino), H.S., and T.U.; formal analysis, T.S.; investigation,
T.S.; resources, T.S., K.H., R.T., Y.H., N.T., M.S. and H.E.; data curation, H.S.; writing—original draft
preparation, T.S.; writing—review and editing, T.U.; visualization, H.K. (Hiroki Kamino); supervision,
H.S.; project administration, Y.N.; funding acquisition, T.U. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Nippon Kayaku Co., Ltd. (to T.S.) and Shimane prefecture (to T.U.). The funders had no role in the design of the study; in the collection, analyses, orinterpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Institutional Review Board Statement: The study was conducted in accordance with the Declarationof Helsinki and approved by the Ethics Committee of MEDIKS (Sapporo, Japan) with approval no.
MX-4150FN-151448. The date of approval was 15 September 2021.
Informed Consent Statement: Informed consent was obtained from all individuals involved in
the study.
Data Availability Statement: The data presented in this study are available in the article.
Conflicts of Interest: T.U. is employed by Shimane University and is currently a co-founder andChief Medical and Scientific Officer of mAbProtein, a biotech company focusing on the development
and commercial utilization of mAbs for inflammation research, diagnosis, and treatment. All theother authors have declared no conflict of interest. The potential conflict of interest by T.U. does notalter the authors’ adherence to all the journal’s policies on sharing data and materials, as detailed
online in the guide for authors.

References
1.
2.

3.

Polack, F.P.; Thomas, S.J.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Perez, J.L.; Marc, G.P.; Moreira, E.D.; Zerbini, C.; et al.
Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N. Engl. J. Med. 2020, 383, 2603–2615. [CrossRef] [PubMed]
Kageyama, T.; Ikeda, K.; Tanaka, S.; Tanigtiuchi, T.; Igari, H.; Onouchi, Y.; Kaneda, A.; Matsushita, K.; Matsushita, K.; Nakada,T.A.; et al. Antibody responses to BNT162b2 mRNA COVID-19 vaccine and their predictors among healthcare workers in a
tertiary referral hospital in Japan. Clin. Microbiol. Infect. 2021, 27, 1861.e1–1861.e5. [CrossRef] [PubMed]
Maeda, K.; Amano, M.; Uemura, Y.; Tsuchiya, K.; Matsushima, T.; Noda, K.; Shimizu, Y.; Fujiwara, A.; Takamatsu, Y.; Ichikawa,Y.; et al. Correlates of neutralizing/SARS-CoV-2-S1-binding antibody response with adverse effects and immune kinetics in
BNT162b2-vaccinated individuals. Sci. Rep. 2021, 11, 22848. [CrossRef] [PubMed]

Infect. Dis. Rep. 2023, 15

4.

5.

6.
7.

8.

9.

10.

11.

12.

13.

14.
15.
16.

17.

18.
19.

33

Naaber, P.; Tserel, L.; Kangro, K.; Sepp, E.; Jürjenson, V.; Adamson, A.; Haljasmägi, L.; Rumm, A.P.; Maruste, R.; Kärner, J.; et al.
Dynamics of antibody response to BNT162b2 vaccine after six months: A longitudinal prospective study. Lancet Reg. Health Eur.
2021, 10, 100208. [CrossRef] [PubMed]Ðaković Rode, O.; Bodulić, K.; Zember, S.; Cetinić Balent, N.; Novokmet, A.; Čulo, M.; Rašić, Ž.; Mikulić, R.; Markotić, A. Declineof anti-SARS-CoV-2 IgG antibody levels 6 months after complete BNT162b2 vaccination in healthcare workers to levels observed
following the first vaccine dose. Vaccines 2022, 10, 153. [CrossRef] [PubMed]
Israel, A.; Merzon, E.; Schäffer, A.A.; Shenhar, Y.; Green, I.; Golan-Cohen, A.; Ruppin, E.; Magen, E.; Vinker, S. Elapsed time since
BNT162b2 vaccine and risk of SARS-CoV-2 infection in a large cohort. medRxiv 2021. [CrossRef]
Nachtigall, I.; Bonsignore, M.; Hohenstein, S.; Bollmann, A.; Günther, R.; Kodde, C.; Englisch, M.; Ahmad-Nejad, P.; Schröder, A.;
Glenz, C.; et al. Effect of gender, age and vaccine on reactogenicity and incapacity to work after COVID-19 vaccination: A survey
among health care workers. BMC Infect Dis. 2022, 22, 291. [CrossRef] [PubMed]
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 coronavirus disease 2019 vaccination. Clin. Infect. Dis. 2021,
73, 2065–2072. [CrossRef] [PubMed]
Ferri, C.; Ursini, F.; Gragnani, L.; Raimondo, V.; Giuggioli, D.; Foti, R.; Caminiti, M.; Olivo, D.; Cuomo, G.; Visentini, M.; et al.
Impaired immunogenicity to COVID-19 vaccines in autoimmune systemic diseases. High prevalence of non-response in different
patients’ subgroups. J. Autoimmun. 2021, 125, 102744. [CrossRef] [PubMed]
Furer, V.; Eviatar, T.; Zisman, D.; Peleg, H.; Paran, D.; Levartovsky, D.; Zisapel, M.; Elalouf, O.; Kaufman, I.; Meidan, R.; et al.
Immunogenicity and safety of the BNT162b2 mRNA COVID-19 vaccine in adult patients with autoimmune inflammatory
rheumatic diseases and in the general population: A multicentre study. Ann. Rheum. Dis. 2021, 80, 1330–1338. [CrossRef][PubMed]Izmirly, P.M.; Kim, M.Y.; Samanovic, M.; Fernandez-Ruiz, R.; Ohana, S.; Deonaraine, K.K.; Engel, A.J.; Masson, M.; Xie, X.;
Cornelius, A.R.; et al. Evaluation of immune response and disease status in systemic lupus erythematosus patients following
SARS-CoV-2 vaccination. Arthritis. Rheumatol. 2022, 74, 284–294. [CrossRef] [PubMed]
Zeng, C.; Evans, J.P.; Reisinger, S.; Woyach, J.; Liscynesky, C.; El Boghdadly, Z.E.; Rubinstein, M.P.; Chakravarthy, K.; Saif, L.; Oltz,
E.M.; et al. Impaired neutralizing antibody response to COVID-19 mRNA vaccines in cancer patients. Cell Biosci. 2021, 11, 197.
[CrossRef] [PubMed]
Demaret, J.; Corroyer-Simovic, B.; Alidjinou, E.K.; Goffard, A.; Trauet, J.; Miczek, S.; Vuotto, F.; Dendooven, A.; Huvent-Grelle, D.;
Podvin, J.; et al. Impaired functional T-cell response to SARS-CoV-2 after two doses of BNT162b2 mRNA vaccine in older people.
Front. Immunol. 2021, 12, 778679. [CrossRef] [PubMed]
Sahin, U.; Muik, A.; Derhovanessian, E.; Vogler, I.; Kranz, L.M.; Vormehr, M.; Baum, A.; Pascal, K.; Quandt, J.; Maurus, D.; et al.
COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses. Nature 2020, 586, 594–599. [CrossRef] [PubMed]
Morell, A.; Terry, W.D.; Waldmann, T.A. Metabolic properties of IgG subclasses in man. J. Clin. Investig. 1970, 49, 673–680.
[CrossRef] [PubMed]
Dan, J.M.; Mateus, J.; Kato, Y.; Hastie, K.M.; Yu, E.D.; Faliti, C.E.; Grifoni, A.; Ramirez, S.I.; Ramirez, S.I.; Ramirez, S.I.; et al.
Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 2021, 371, eabf4063. [CrossRef][PubMed]Wajnberg, A.; Amanat, F.; Firpo, A.; Altman, D.R.; Bailey, M.J.; Mansour, M.; McMahon, M.; Meade, P.; Mendu, D.R.; Muellers,K.; et al. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. Science 2020, 370, 1227–1230. [CrossRef][PubMed]Chen, Y.; Klein, S.L.; Garibaldi, B.T.; Li, H.; Wu, C.; Osevala, N.M.; Li, T.; Margolick, J.B.; Pawelec, G.; Leng, S.X. Aging in
COVID-19: Vulnerability, immunity and intervention. Ageing Res. Rev. 2021, 65, 101205. [CrossRef] [PubMed]
Deepak, P.; Kim, W.; Paley, M.A.; Yang, M.; Carvidi, A.B.; El-Qunni, A.A.; Haile, A.; Huang, K.; Kinnett, B.; Liebeskind, M.J.; et al.
Glucocorticoids and B cell depleting agents substantially impair immunogenicity of mRNA vaccines to SARS-CoV-2. medRxiv
2021. [CrossRef]

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