viruses
ArticleAcquisition of Humoral Immune Responses in ConvalescentJapanese People with SARS-CoV-2 (COVID-19) Infection in 2021
Koshiro Monzen 1, *, Takanori Watanabe 2 , Toshihiro Okabe 3 , Hisakuni Sekino 4 , Hironori Nakagami 5
and Ryuichi Morishita 6
1
2
3
4
5

6

*

Citation: Monzen, K.; Watanabe, T.;
Okabe, T.; Sekino, H.; Nakagami, H.;
Morishita, R. Acquisition of Humoral
Immune Responses in ConvalescentJapanese People with SARS-CoV-2(COVID-19) Infection in 2021. Viruses
2023, 15, 1842. https://doi.org/

Shinjuku Tsurukame Clinic, 2-11-15 Yoyogi, Shibuya-ku, Tokyo 151-0053, Japan
Uehonmachi Watanabe Clinic, 1-15 Uenomiya-cho, Tennoji-ku, Osaka 543-0037, Osaka, Japan
Ueda Hospital, 1-7-1 Inazu-cho, Toyonaka 561-0854, Osaka, Japan
Sekino Hospital, 3-28-3 Ikebukuro, Toshima-ku, Tokyo 171-0014, Japan
Department of Health Development and Medicine, Graduate School of Medicine, Osaka University,
2-2 Yamada-oka, Suita 565-0871, Osaka, Japan
Department of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka,
Suita 565-0871, Osaka, Japan
Correspondence: monzen@tsurukamekai.jp

Abstract: We investigated humoral immune responses in 222 unvaccinated Japanese people after
recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2021.
Anti-spike-protein IgG antibody levels and neutralizing antibody titers were measured in serum
samples obtained within 20–180 days after diagnosis. The geometric mean of antibody titers was
1555 ELU/mL (95% confidence interval (CI) = 1257–1923), and the neutralizing activity (50% inhibitory dilution) was 253 (95% CI = 204–313). The antibody titer and neutralizing activity bothincreased with increasing disease severity, and both values were approximately fourfold higher for
hospitalized patients than for non-hospitalized patients. However, these differences were smallerin older patients. The humoral immune response, which increased with increasing disease severity,
gradually decreased over time after SARS-CoV-2 infection. Most patients with mild or moderate
symptoms sustained neutralizing activity for up to 180 days after the infection; the decay of theneutralizing activity in the asymptomatic patients was rather faster than in the other groups. Around
11.7% (26/222) of patients had very low neutralizing activity, and half of these were aged in their
20s. Our study’s results show the importance of measuring the neutralizing activity to confirm theimmune status and to estimate the timing of vaccines.
Keywords: COVID-19; SARS-CoV-2; convalescent serum; neutralizing antibody

10.3390/v15091842
Academic Editors: Luciana Jesus
Costa and Amilcar Tanuri

1. Introduction

Received: 28 July 2023

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acuterespiratory syndrome coronavirus 2 (SARS-CoV-2), which was first identified in 2019 in
Wuhan, China [1]. The outbreak of COVID-19 began with “pneumonia of unknown cause”
in Wuhan, Hubei Province, in December 2019, followed by the international spread ofthe disease, mainly in China. The World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC) on 30 January 2020, and as of
18 March 2021, the WHO had reported 120,383,919 cases and 2,664,386 deaths worldwide,with 223 countries/regions having confirmed cases. Multiple coronavirus variants (e.g.,
Alpha, Beta, Gamma, Delta, and Omicron) have been discovered to date, and they havespread globally (WHO).
It remained difficult to control SARS-CoV-2 transmission during pandemic waves
because of the large number of people with asymptomatic infection, who had similar viralloads to those of symptomatic patients, as well as viral shedding in symptomatic patients
before symptom onset. In Japan, seven SARS-CoV-2 epidemic waves were encountered

Revised: 24 August 2023
Accepted: 28 August 2023
Published: 30 August 2023

Copyright: © 2023 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/).

Viruses 2023, 15, 1842. https://doi.org/10.3390/v15091842

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

Viruses 2023, 15, 1842

2 of 12through the middle of 2022. Each wave consisted of a sharp surge and subsequent decline in
new infection cases. Some reports described the acquisition of humoral immune responses
after recovery from SARS-CoV-2 infection in Japanese patients [2,3]; D614G-mutationcarrying variants were the primary causes of COVID-19 [4].
Many important reports on the relationship between antibody production and patient
background have been accumulated. In general, neutralizing antibody activities in patientswith COVID-19 vary widely from patient to patient; peak neutralizing antibody activities,
however, are known to increase in proportion to the severity of the disease [5]. However,
the well-balanced and coordinated function of antibody-producing B cells and T cells is
important for biological defense during acute infection with novel coronaviruses [6], andthe importance of the natural immune system has been pointed out, which contributes toan asymptomatic recovery despite the low production of antibodies [7]. Thus, a complex
network of collaboration and communication among cells functioning to eliminate the virus
should be considered; however, the information regarding antibody levels and activity
analyzed by patient background and the decay of neutralizing activity remains insufficient.
To manage the inoculation strategy for the available coronavirus vaccines, it is considered to be important for Japanese medical personnel to know the naturally acquired
post-SARS-CoV-2 infection immunity status of patients and compare it with coronavirusvaccine-derived immunity. In this study, we evaluated the anti-spike and neutralizing
antibody levels of patients infected with SARS-CoV-2 between January 2021 and September
2021. During this period, the Alpha, Delta, and D614G-mutation-carrying variants were
the primary causes of COVID-19 [4]. The objective of this study was to obtain information
for the development of new prophylactic vaccines and new treatments against COVID-19
in Japan.
2. Materials and Methods
2.1. Study Samples
2.1.1. Inclusion Criteria
Serum samples from non-hospitalized individuals or hospitalized individuals who hadrecuperated from previous SARS-CoV-2 infection, satisfying all of the following inclusion
criteria, were selected in this observational study:
1.
2.

3.
4.

5.

Samples from study subjects from whom written voluntary informed consent toparticipate in this study was obtained.
Samples from study subjects who gave consent for the principal investigator, etc.,
to collect information on the diagnosis, treatment, etc., from the medical institution,
health center, etc., where the SARS-CoV-2 infection was diagnosed/treated.
Samples from study subjects aged 20 years or older at the time of informed consent.
Samples from study subjects who had recuperated from infection after testing positive
for SARS-CoV-2 diagnosed through nucleic acid detection or antigen testing, and whotested negative on nucleic acid detection or antigen tests, or archival samples fromindividuals who had recuperated and were similar to those mentioned above.
Samples from 20 to 180 days after testing positive for SARS-CoV-2.

2.1.2. Exclusion Criteria
Samples were excluded from the analysis if met any of the following criteria:
1.
2.
3.
4.

Samples from individuals who had not recuperated from SARS-CoV-2 infection.
Samples from recipients of prophylactic COVID-19 vaccines (including products
in development).
Samples for which there was a request to withdraw consent.
Samples considered ineligible by the principal investigator or sub-investigator.

Viruses 2023, 15, 1842

3 of 122.2. Study Procedure
The SARS-CoV-2 infection was confirmed by nucleic acid detection or antigen testing.
Samples from study subjects who had recuperated from infection or archival samples fromindividuals who had recuperated were transported to a testing facility in order to measureneutralizing activity against the pseudovirus of SARS-CoV-2 and SARS-CoV-2 spike (S)glycoprotein-specific antibody titers. For a sample collected from a study subject, a nucleic
acid detection test or antigen test was performed again to confirm the SARS-CoV-2 infection
status at the time of sample collection. If negative results were obtained, the data were
included in the analysis set of this study, and if positive results were obtained, the data
were excluded from the analysis set. Even if archival samples were used, data from samples
with negative results were included in the analysis set.
The following information was entered into the case report form for each study subject:anonymized study ID, age, sex, onset date, date of definitive diagnosis, definitive diagnosis
result (nucleic acid detection or antigen test), non-hospitalization/hospitalization, severityof SARS-CoV-2 infection, result of assessment of recuperation from infection (nucleic aciddetection or antigen test), and date of sample collection. The severity was classified into fivelevels as follows: asymptomatic, mild (no respiratory symptoms or cough only, without
dyspnea; SpO2 ≥ 96%; no evidence of pneumonia in any case), moderate I (no respiratory
failure, pneumonia findings, or dyspnea; 93% < SpO2 < 96%), moderate II (respiratory
failure requiring supplemental oxygen; SpO2 ≤ 93%), and severe (admitted to intensive
care unit or requiring a ventilator).
In this study, personal information was managed using an enrollment number uniqueto each study subject, assigned for anonymization. A correspondence table of enrollment
numbers was appropriately managed by the department in charge at the study institution
providing the biological samples. In addition, materials and correspondence tables containing other personal information collected in this study were managed appropriately,in compliance with the management methods specified at the institution providing thebiological samples, to protect personal information. When sharing the study’s results withinstitutions providing biological samples, data were handled only with the anonymized
study ID assigned for this study. The only information to be provided to the collaborative
research institution was the anonymized study ID, measurement data, and information
without personal identification, and information that could identify a particular individual
was not provided in order to prevent any possibility of risk or disadvantage associatedwith the leakage of information about the study subjects.
2.3. Sample Analyses
SARS-CoV-2 anti-spike IgG concentrations were measured at Nexelis (Laval, QC,
Canada) using S-ELISA, and neutralizing activity (50% inhibitory dilution: ID50 ) was
determined using the PhenoSense SARS-CoV-2 neutralizing antibody assay (PNA) at
Labcorp (Indianapolis, IN, USA). The analytical methods of S-ELISA and PNA werevalidated by Nexelis and Labcorp, respectively.
The SARS-CoV-2 pre-spike recombinant antigen was adsorbed onto a 96-well microplate. Following incubation, the microplate was washed to remove unbound SARS-CoV-2
pre-spike recombinant antigen and blocked to prevent non-specific binding. Standards,
controls, and sample dilutions were incubated in the coated microplate, in which antiSARS-CoV-2 pre-spike IgG-specific antibodies (primary antibodies) bound to the coatedantigen. Following incubation, the microplate was washed to remove unbound primary
antibodies. Primary antibodies were detected with the addition of the anti-human IgG
antibody (secondary antibody) conjugated to peroxidase. After incubation, the microplate
was washed to remove unbound secondary antibodies. The peroxidase substrate solution,
tetramethylbenzidine (TMB), was added to the microplate, and a colored product wasdeveloped that was proportional to the amount of anti-SARS-CoV-2 pre-spike IgG antibodies present in the serum sample. Then, 2N H2SO4 was added to stop the colorimetric
reaction. The absorbance of each well was measured using a microplate spectrophotometer

Viruses 2023, 15, 1842

4 of 12reader at a specific wavelength (450/620 nm). Antibody concentrations were calculated
for each control and sample dilution by interpolation of the OD values on the 4-parameter
logistic (4-PL) standard curve and adjusted according to their corresponding dilution factor.
The final concentrations of controls and samples were then determined by calculating thegeometric mean of all adjusted concentrations (for the given control or sample) obtained
within the interpolation range of the standard curve. The mean absolute percentage ofthe relative error calculated from all standard points had to be 15.0% or less. Sample andcontrol concentrations were expressed as ELISA laboratory units per milliliter (ELU/mL).
The measurement of neutralizing activity using PNA was performed by generating
HIV-1 pseudovirions that expressed the SARS-CoV-2 spike protein. The reporter pseudovirus was prepared by co-transfecting HEK293 producer cells with an HIV-1 genomic
vector and a SARS-CoV-2 envelope expression vector. Neutralizing antibody activity wasmeasured by assessing the inhibition of luciferase activity in HEK293 target cells transiently
expressing the ACE2 receptor following pre-incubation of the pseudovirions with theserum specimen. A serial dilution of the test serum specimen was incubated with a reporter
pseudovirus to generate an inhibition curve that enabled the determination of an ID50 foreach sample. Luminescence as an index of capability of inhibiting the virus from replicating
and thereby preventing the luciferase by samples was measured as relative luminescentunits (RLU). The acceptable limit of intra-assay and inter-assay precision was set <30% ID50
CV.
The detection limit for ELISA titers (EUL/mL) and neutralizing activity (ID50 ) was
50 and 40, respectively. Accordingly, a value of 25 (half the minimum required dilution) for
ELISA and a value of 20 (half the minimum required dilution) for PNA were assigned tosamples below the cutoff point.
2.4. Statistical Analyses
Frequency tables, number of specimens, and percentages were calculated from the categorical data. Continuous variables were expressed as the mean, median, minimum/maximum,and interquartile range. Antibody titers and neutralizing activity were stratified by age,sex, and disease severity. The geometric mean and 95% confidence interval were calculated
for both S-ELISA and PNA data from eligible participants. Data analysis was performed by
EPS Corporation (Tokyo, Japan), using SAS® 9.4 (SAS Institute Inc., Cary, NC, USA).
2.5. Sample Size
In a similar study overseas [8], the correlation between the severity of COVID-19 andneutralizing antibody activities was evaluated using 32 samples from non-hospitalized
individuals and 40 samples from hospitalized individuals. In this study, the sample sizeof at least 50 samples from non-hospitalized individuals and at least 50 samples from
hospitalized individuals was set based on the number of individuals who had recuperated
from previous SARS-CoV-2 infection that could be accrued by December 31, 2021 (the end
date of the study period), using this overseas study report as a reference.
3. Results
3.1. Clinical Characteristics
A total of 222 patients were eligible. Of these, 72 patients were hospitalized, and
136 patients (61.3%) were male. The gender balance was similar in the non-hospitalized
and hospitalized groups. The mean age of the entire cohort was 40.0 years, and the mean
ages of the non-hospitalized and hospitalized groups were 36.6 and 47.1 years, respectively,
suggesting that older patients were more likely to require hospitalization. Concerning theseverity of SARS-CoV-2 infection, 8 patients (3.6%) were asymptomatic, 146 patients (65.8%)
had mild symptoms, 39 patients (17.6%) had moderate I symptoms, and 29 patients (13.1%)
had moderate II symptoms. The majority of non-hospitalized patients had mild symptoms(94.0%), whereas most hospitalized patients had moderate I or moderate II severity (52.8%
and 40.3%, respectively; Table 1).

Viruses 2023, 15, 1842

5 of 12

Table 1. Patient characteristics.
Non-Hospitalized
N = 150n (%)

Hospitalized
N = 72n (%)

Total
N = 222
n (%)

Male

92 (61.3)

44 (61.1)

136 (61.3)

Female58 (38.7)

28 (38.9)

86 (38.7)

n

150

72

222

Mean (SD)36.6 (13.1)

47.1 (11.7)

40.0 (13.5)

Median

33.0

49.0

40.0

Min, max

20, 7820, 7920, 7920 to <40 *92 (61.3)

17 (23.6)

109 (49.1)

40 to <60

52 (34.7)

47 (65.3)

99 (44.6)≥60

6 (4.0)8 (11.1)

14 (6.3)

Asymptomatic

8 (5.3)08 (3.6)

Mild

141 (94.0)

5 (6.9)146 (65.8)

Moderate I

1 (0.7)38 (52.8)

39 (17.6)

Moderate II0

29 (40.3)

29 (13.1)

Severe

0

0

0

Sex

Age (years)

Severity of
SARS-CoV-2 infection* Age of 20 to <30: total N = 64 (57 non-hospitalized, 7 hospitalized).

3.2. Immunogenicity Assessments
As S-ELISA result was deemed invalid for one patient, so the numbers of test resultsfor S-ELISA and PNA were 221 and 222, respectively.
The geometric mean of anti-spike antibody titers and neutralizing activity were1555 ELU/mL and 253 ID50 (the reciprocal serum dilution corresponding to 50% neutralization), respectively, for eligible patients (Table 2). There were no differences in either
parameter according to sex. Both the anti-spike antibody titer and the neutralizing activity were approximately fourfold higher in hospitalized patients than in non-hospitalized
patients, excluding patients older than 60 years. Both anti-spike antibody titers and neutralizing activity in the age group of ≥30 to <40 years was larger than that in the othertwo groups. No distinct differences in anti-spike antibody titer were observed between
hospitalized and non-hospitalized patients in the age group of >60 years, but older hospitalized patients displayed relatively lower neutralizing activity. Both anti-spike antibody
titers and neutralizing activity increased with disease severity (Table 2).
The distribution of anti-spike antibody titers by each patient is shown in Figure 1A.
Seven patients (3.4%) had antibody titers lower than the detection limit (1.7; log10 ELISA
titer), in all of whom the neutralizing activity was below the detection limit (1.6; log10
ID50 ), although COVID-19 had been confirmed by polymerase chain reaction or antigen
test. Including these 7 patients, anti-spike antibody titers were low in 8 patients (3.6%)(log10 ELISA titer < 2), low–medium in 21 patients (9.5%) (2 ≤ log10 ELISA titer < 2.5),
medium in 69 patients (31.2%) (2.5 ≤ log10 ELISA titer < 3), high–medium in 49 patients(22.2%) (3 ≤ log10 ELISA titer < 3.5), and high in 51 patients (23.1%) (log10 ELISA titer ≥ 3.5).
Twenty-three patients developed extremely high antibody titers (log10 ELISA titer > 4). The
gender of these 23 patients was 17 males and 6 females. Two patients aged ≥ 60 years were
included in this population.

Viruses 2023, 15, 1842

6 of 12

Table 2. Antibody titers and neutralizing activity stratified by patient characteristics between nonhospitalized and hospitalized patients.
Antibody Titer (ELU/mL): S-ELISA
Non-Hospitalized

Hospitalized

Total

Non-Hospitalized

Hospitalized

Total

941

4391

1555

166

607

253

(753, 1176)(3052, 6317)(1257, 1923)(132, 209)

(415, 888)

(204, 313)

N = 149

N = 72N = 221N = 150

N = 72N = 222977

4062

1549.55

182

638

273

(727, 1312)(2449, 6737)(1172, 2047)(133, 247)

(384, 1059)(206, 361)

N = 92

N = 44

N = 136

N = 92

N = 44

N = 136

886

4963

1563

144

562

224

(627, 1253)(2923, 8425)(1119, 2183)(101, 204)

(306, 1030)(161, 312)

N = 58N = 28N = 85N = 58N = 28N = 86

881

2932

1065

150

375

173

(696, 1117)(1376, 6245)(837, 1356)(111, 202)

(172, 817)

(131, 229)

N = 91

N = 17N = 108

N = 92

N = 17N = 109

1050

6209

2442

199

886

405

(667, 1652)(4173, 9240)(1725, 3456)(134, 295)

(561, 1399)(291, 563)

N = 52

N = 47

N = 99N = 52

N = 47

N = 99

983

1353

1180

159

183

172

(80, 12,015)(228, 8006)(339, 4099)(24, 1032)(52, 636)(70, 423)

N=6

N=8

N = 14N=6

N=8N = 14

319

-

319

105

-

105

(116, 877)-

(116, 877)

(31, 353)-(31, 353)

N=8

N=0

N=8

N=8

N=0

N=8

992

4221

1043

169

610

176

(790, 1246)(77,
229,822)(819, 1327)(133, 214)(8, 43,608)(137, 227)

N = 140

N=5N = 145

N = 141

N=5N = 146

3421

3241

3245

591

521

523

-

(1877, 5595)(1908, 5521)-(302, 899)

(308, 888)

N=1

N = 38N = 39N=1

N = 38N = 39

6582

6582

-

741

741

-

(4840, 8950)(4840, 8950)-(484, 1132)(484, 1132)

N=0

N = 29N = 29N=0

N = 29N = 29All

GenderMale

FemaleAge

20 to <40(years)40 to <60

≥60

Severity

Asymptomatic

Mild

Moderate
I

Neutralizing Activity (ID50 ): PNA

Moderate
II

Antibody titers and neutralizing activity are expressed as the geometric mean. The 95% confidence intervals arepresented within brackets. One patient was excluded from the analysis because of an invalid ELISA result.

VirusesViruses
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x 15, 1842
2023,

7 of 127 of 12

FigureDistributionsof
ofanti-spikeanti-spike antibody
andneutralizing
antibody
activity(B) by(B) by
Figure1. 1.
Distributions
antibodytiters
titers(A)(A)
andneutralizing
antibody
activity
patient.
Sevenpatientshad
hadanti-spikeanti-spike antibody
antibody titers
limit
(1.7;
loglog
ELISA
patient.
Sevenpatients
titersbelow
belowthethedetection
detection
limit
(1.7;
10 10 ELISA tititer),twenty-six
patientshad
hadneutralizing
neutralizing antibody
activity
below
the detection
limit (1.6;
ter),
andandtwenty-six
patients
antibody
activity
below
the detection
limitlog(1.6;
10 log10). Forconvenience,thethefollowing
following criteria
forforthethelevelsof antibody
activity
and and
ID50ID). 50For
convenience,
criteriawere
wereapplied
appliedlevelsof antibody
activityneutralizing
antibody
low(log(log
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low–medium(2 10≤ ID
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activity: low
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1010ID50
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or
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ID50 ortiter
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titer
10ELISA
titerELISA<3.5),titerand<high(loghigh
10 ID(log
50 or
≥3.5)
. ≥ 3.5).

The distribution of neutralizing activity, in ascending order, is shown in Figure 1B.
The distributionof neutralizing
in ascendingis shown
in Figure
Twenty-six
patients had
neutralizing activity,
activity lower
than the order,
detection
limit (1.6;log10 1B.
Twenty-six
had neutralizing
than the20detection
limitof(1.6;
ID50 ). Of patientsthese 26 patients,
16 patientsactivity(61.5%)lower
were between
and 39 years
age. log10
ID50). Of thisthese
26grouppatients,
patients(61.5%)
between
20 and39included
years of in
age.
When
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was16
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dividedintowere
two groups,
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were
theWhenage group,and 3/16
were included
the 30–39
ageweregroup.
Six were
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13/16
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and fourincludedthe overyears
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Bytheseverity,group,
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Six 60were
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40–59 age2
patients
wereasymptomatic,
22
mild,
and
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moderate
I.
The
antibody
titersinthis weregroup, and four were included in the over 60 years group. By severity, 2 patients
population were low in 8 patients, including 7 patients with titers below the detection
asymptomatic, 22 mild, and 2 moderate I. The antibody titers in this population were low
limit of ELISA, low–medium in 12 patients (2 ≤ log10 ELISA titer < 2.5), and medium inin 8 patients, including 7 patients with titers below
the detection limit of ELISA, low–
6 patients (2.5 ≤ log10 ELISA titer < 3).
mediumin
12
patients(2≤log
10 ELISA titer <2.5), and medium in 6 patients (2.5 ≤ log10
Neutralizing activities were low in 39 patients (17.6%) (log10 ID 50 < 2), low–medium in
ELISA
titer
69 patients<3).
(31.1%) (2 ≤ log10 ID50 < 2.5), medium in 46 patients (20.7%) (2.5 ≤ log10 ID50 < 3),
10 in
ID950patients< 2), low–medium
Neutralizinginactivities
were
low(3in(17.6%)
high–medium
28 patients
(13%)
≤ 39
logpatients
and(loghigh(4.1%)
10 ID50 < 3.5),(logpatients3.5). Fourthewho developed
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10 five
ID50patients< 2.5), medium
in 46 patients(20.7%)(2.5 ≤ log10 ID50 <in 69(31.1%)(2 ≤oflog
10 ID50 ≥(log10 ID50
were male.
3), activity
high–mediumin >284) patients(13%) (3 ≤ log10 ID50 < 3.5), and high in 9 patients (4.1%)
The
anti-spike
antibody
titersand neutralizing
activities extremely
displayed ahighgoodneutralizing
correlation ac(log10 ID50 ≥ 3.5). Four of the five patients
who developedin both the non-hospitalized and hospitalized groups, and the correlation coefficient in all
tivity (log10 ID50 > 4) were male.
patients was 0.84 (Figure 2). Neutralizing activities gradually decreased over time after
The anti-spike antibody titers and neutralizing activities displayed a good correlaSARS-CoV-2 infection. In asymptomatic patients, serum neutralizing activity tended totion
in both thenon-hospitalized
andnothospitalized
andbetween
the correlationcoeﬃcientdisappear
earlier;
however, we could
find a clear groups,
relationshipthe severityof
in allpatients
was
0.84(Figure2).
Neutralizing
activities
gradually
decreasedover time
SARS-CoV-2 infection and the disappearance of antibody activities. Most patients withafter
infection.had
In sustained
asymptomaticpatients,
serum
neutralizing
activity
mildSARS-CoV-2
or moderate symptoms
neutralizing
activity
up to 180
days after the
SARS-CoV-2
infection(Figure3).
tendedto disappear
earlier;
however,
we could not find a clear relationship between theseverity of SARS-CoV-2 infection and the disappearance of antibody activities. Most patients with mild or moderate symptoms had sustained neutralizing activity up to 180
days after the SARS-CoV-2 infection (Figure 3).

Viruses 2021, 13, x

Viruses 2021,
2023, 13,
15, x
1842

8 of 128 8ofof 12
12

FigureRelationshipbetween
betweenanti-spike
antibody
and neutralizing
correlation
Figure 2.2. Relationship
Relationshipbetweenanti-spike
antibody
titerstiters
and neutralizing
neutralizing
activity;activity;
correlation
Figureanti-spike
antibody
titersandactivity;correlationcoeﬃcientwas
calculated
excludingone
patient
withinvalid
ELISA
data.
coeﬃcientcoefficient waswas calculated
calculated excluding
excluding one
one patientpatient with invalid ELISA data.

Figure 3. Neutralizing activity by days after infection. X-axis: days after the diagnosis of
COVID-19. Most patients with mild or moderate symptoms had sustained neutralizing activity up
Figure3.3.Neutralizing
activityby days
infection.
X-axis:days X-axis:
after
the diagnosis
of COVID-19.
to180 days
after infection.
There
were
26dayspatients
withneutralizing
activitybelow
thedetection
FigureNeutralizing
activity
byafter
after
infection.
days
after
the
diagnosis oflimit,
including
2 asymptomatic,
22 mild,
and 2 moderate
I cases.
Most
patients
with
mild
or
moderate
symptoms
hadsustained
neutralizing
activityupto
180 days
COVID-19. Most patients with mild or moderate symptoms had sustained neutralizing
activity up
after
There
were 26 patientswith neutralizing
below the detection
limit,
includingto180infection.
days after
infection.
There were
26 patientsactivity
with neutralizing
activity
below
the detection
4.
Discussion 22 mild, and 2 moderate I cases.
2 asymptomatic,
limit,
including 2 asymptomatic, 22 mild, and 2 moderate I cases.

In this cross-sectional study, we quantified anti-spike and neutralizing antibody
levelsin patients who contracted COVID-19 between January and September 2021. Ac4. Discussion
cordingly, it is assumed that the dominant strains were the D614G variant from January

In this cross-sectional study, we quantified anti-spike and neutralizing antibody
levels in patients who contracted COVID-19 between January and September 2021. Accordingly, it is assumed that the dominant strains were the D614G variant from JanuaryViruses 2023, 15, 1842

9 of 12

4. Discussion
In this cross-sectional study, we quantified anti-spike and neutralizing antibody levels
in patients who contracted COVID-19 between January and September 2021. Accordingly,
it is assumed that the dominant strains were the D614G variant from January to March,
the Alpha strain from April to June, and the Delta strain from July to September [4].
In total, 96.5% (141 of 146) of patients with mild respiratory symptoms or high oxygen
saturation did not require hospitalization, whereas 97% (38 of 39) of patients with moderate
dyspnea or pneumonia required hospitalization, suggesting that medical care was provided
appropriately during the pandemic in Japan.
It has been reported that male sex, older age, and hospitalization are associated withthe anti-spike IgG response [9,10]. In the analysis stratified by age, the antibody titers inour study were lower in patients aged 60 years and older, and antibody levels did not
differ between hospitalized and non-hospitalized patients in this age group, illustratingthat antibodies were not produced according to disease severity and suggesting an insufficient immune response to new pathogens in older patients. Apart from this, age was
identified as a negative independent variable for serum anti-SARS-CoV-2S antibody levels
after immunization with mRNA-based COVID-19 vaccines, based on data from more than2000 people [11]. Anti-spike antibody titers and neutralizing activity did not differ according to sex in this study. This result is consistent with those reported by Trinite et al. [8],who found that the plateau of neutralizing activity was similar between men and women,even though the maximum titers of neutralizing antibodies significantly differed. It is
likely that there is no apparent gender difference in terms of antibody production [9,10].
However, Scully et al. reported that males are associated with a greater risk of more severe
COVID-19 outcomes [12].
In this study, when patients with COVID-19 were classified by disease severity, antibody titers and neutralizing activity were low in asymptomatic and mildly ill patients,
whereas they were high in patients with moderate disease severity. These results wereconsistent with those reports showing that severely ill patients with COVID-19 have higher
anti-spike antibody levels, as well as the production of more potent neutralizing antibodies [8,13]. However, it is noteworthy that Trinité et al. reported no correlation between
neutralizing capacity and length of hospitalization, indicating the possibility that thepresence of neutralizing antibodies is not a determinant for the disease resolution, i.e.,a contradictory situation in which neutralizing activities are not associated with clinicalbenefit [8]. Furthermore, it is suggested that while antibody production plays an importantrole in the elimination of the SARS-CoV-2 virus, the well-balanced function of both CD4+
T cells essential for antibody production and memory-B-cell formation, and CD8+ T cells
providing protection against antigens, is more important for preventing aggravation [6]Our analysis illustrated that some patients have neutralizing activity of less than
1.6 log10 ID50 despite moderate antibody levels, suggestive of the production of nonneutralizing antibodies, as well as a risk of reinfection. The neutralizing activity of each IgGfraction and the amount of SARS-CoV-2-binding antibodies in serum/plasma obtained at
multiple timepoints were determined in 43 patients [14], and 16 patients with considerable
antibody titers had no neutralizing activity during the observation periods. It is curiousthat the cohort with these characteristics mainly included patients with mild severity, andthis finding was common to both studies. Further investigation regarding risk factors forthe production of antibodies with insufficient activity is required in the future. Antibody
levels below the detection limit were observed in younger patients in our study. The reason
that this type of patient was exclusively distributed in this group is uncertain. In general,
if the innate immunity adequately and strongly functioned, the virus could have been
eliminated before the production of antibodies, along with functions for the aggravation of
infection. Moderbacher et al. proposed the important role of higher levels of naïve T cells
for eliminating viruses in the young [6]. In addition, the importance of a robust naturalimmune system has been pointed out, which may contribute to asymptomatic recovery
despite the low production of antibodies in children and young people [7,15].

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Antibody titers in patients with moderate or severe disease persist for a relatively long
period (up to 180 days). Previous reports in Japan have also found that COVID-19 survivors
had sustained neutralizing activity for approximately 6 or 12 months after infection [2,3].
How long the neutralizing activity of antibodies is maintained is important informationfor estimating the risk of reinfection. The antibody decay was analyzed by Khoury et al.,and they extrapolated that the neutralizing activity would drop below the detection limit
around 240 days after outbreak, while the half-life was estimated to be approximately
90 days, from which the model assumed that the decay in neutralizing activity would be
the same regardless of the initial antibody titers [16]. The data concerning the antibody
titers and neutralizing activity necessary for preventing infection are currently limited.
However, after plasma antibodies from monkeys that had recovered from SARS-CoV-2
infection were transfused to other monkeys at various concentrations before challengewith SARS-CoV-2, the antibody titers required for protection, i.e., decreasing the amountof virus in the upper respiratory tract compared to that in the control group, was estimated at approximately 50 pseudovirus neutralizing antibody activity in the blood before
infection [17]. By utilizing the data from seven clinical trials for COVID-19 vaccines andone convalescent study, Khoury et al. reported that the neutralizing activity necessary tohalve the chance of infection is equivalent to about 20% of the mean neutralizing activity ofconvalescent plasma based on a normally distributed model, and about 30% by using a
distribution-free approach [16].
The samples were cross-sectional, and the window for data collection was relatively
tight. The sampled population was considered to be clinically diverse, and its wide age
range is representative of the blood donor population in general clinical practice. However,this study had multiple limitations. Serum samples were not obtained from patientswith severe symptoms in this study; thus, we could not fully analyze the relationshipbetween the levels and activity of antibodies or examine the time course of antibody
activity across patients with COVID-19. The data released by the Japanese government
from three different districts between January and February 2022 illustrate that the rate ofsevere disease among infected people with no history of vaccination remains low (less than
0.5% (145/34,136 patients)) [18]. This might partially support the plausibility of samplingbias in this analysis. In addition, this study contained fewer asymptomatic patients (n = 8)
and elderly patients (n = 14). Furthermore, this study consisted of samples provided by
the patients at a single and arbitrary point (pooled data), rather than from sequential
sampling in each patient. In interpreting the results of this stratified analysis of antibody
titers and neutralizing activities, it is important to note that the time after infection, i.e.,
antibody decay, was not considered; therefore, this could remain as a substantial bias.
The samples were collected from January to September 2021, when different variants
were dominant. This might have caused a bias to the obtained antibody titers, since theantibodies’ affinity/neutralization capacity will vary with different variants. We also could
not eliminate biases such as underlying factors—especially those affecting the immune
system, such as antibody production—as well as alternative confounding effects associatedwith the baseline characteristics used in this analysis, and residual confounders.
5. Conclusions
Overall, this cross-sectional observational study revealed that the production of antispike antibodies with neutralizing activity was sustained for more than 6 months in patients
who were infected with SARS-CoV-2 variants such as the Alpha and Delta variants, as wellas variants carrying the D614G mutation, and also showed that there existed patients who
had very low neutralizing activity (particularly in young people), as well as those who
had low neutralizing activity despite a certain level of antibody titers. The results of thisanalysis illustrate the importance of appropriately measuring neutralizing activity andprovide useful information regarding the management of COVID-19 vaccination.

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Author Contributions: Conceptualization, R.M.; methodology, R.M.; formal analysis, K.M., T.W.,
T.O. and H.S.; investigation, K.M., T.W., T.O. and H.S.; data curation, K.M., T.W., T.O. and H.S.;
writing—original draft preparation, H.N.; writing—review and editing, K.M., T.W., T.O., H.S. and
R.M. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by a Project Promoting Support for Drug Discovery grant(JP21nf0101623h0002) from the Japan Agency for Medical Research and Development.
Institutional Review Board Statement: In the case study, subjects visited the study institution forsamples to be collected, and written consent was obtained using the specified informed consent form.
In cases where archival samples retained at the study institution were used, the sample provider
was notified through an opt-out procedure and, if an intention to withdraw consent was expressed,
the sample was not utilized. The study was conducted according to the “Helsinki Declaration of
the World Medical Congress”. For this study, approval was obtained from the institutional review
board of the representative study institution (Sekino Hospital, Tokyo) prior to conducting the study,
and then an application for approval was submitted to the institutional review board of each study
institution (approval No. and date: 20210618-22S372 at IRB No. 14000050 on 16 July 2001, and 2102 at
Sekino Hospital IRB on 16 July 2001). After approval was obtained at each study institution, the study
was sequentially started. The outline of this study was registered on the public database (UMIN-CTR)
established by the National University Hospital Council of Japan, under the number UMIN000044638,
and disclosed to the public prior to the start of the study.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data for this study will available upon request.
Acknowledgments: We thank Medical Grown Co., Ltd. for scientific editing support and formatting
to Journal style and editorial support.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
ID50
CI
COVID-19
PNA
SARS-CoV-2
S-ELISA
UMIN

50% inhibitory dilution
Confidence interval
Coronavirus disease 2019
PhenoSense SARS-CoV-2 neutralizing antibody assay
Severe acute respiratory syndrome coronavirus 2
SARS-CoV-2 spike enzyme-linked immunosorbent assay
University Hospital Medical Information Network

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