Article

COVID-19 Vaccination in Pregnancy: Pilot Study of Plasma

MicroRNAs Associated with Inflammatory Cytokines after
COVID-19 mRNA Vaccination
Ching-Ju Shen 1 , Yen-Pin Lin 2 , Wei-Chun Chen 2,3,4,5,6 , Mei-Hsiu Cheng 7 , Jun-Jie Hong 7 , Shu-Yu Hu 2 ,
Ching-Fen Shen 8, * and Chao-Min Cheng 2, *

1 Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical
University, Kaohsiung 807, Taiwan; [email protected]
2 Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan;
[email protected] (Y.-P.L.); [email protected] (W.-C.C.); [email protected] (S.-Y.H.)
3 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Chang Gung Memorial
Hospital at Linkou, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
4 Department of Obstetrics and Gynecology, New Taipei City Municipal Tucheng Hospital,
New Taipei City 236, Taiwan
5 International Intercollegiate Ph.D. Program, National Tsing Hua University, Hsinchu 300, Taiwan
6 School of Traditional Chinese Medicine, Chang Gung University, Taoyuan 333, Taiwan
7 Taiwan Business Development Department, Inti Taiwan, Inc., Hsinchu 302, Taiwan;
[email protected] (M.-H.C.); [email protected] (J.-J.H.)
8 Department of Pediatrics, National Cheng Kung University Hospital, College of Medicine,
National Cheng Kung University, Tainan 701, Taiwan
* Correspondence: [email protected] (C.-F.S.); [email protected] (C.-M.C.)

Abstract: Background: The impact of mRNA COVID-19 vaccines on the immunological profiles
of pregnant women remains a crucial area of study. This research aims to explore the specific
immunological changes triggered by these vaccines in this demographic. Methods: In a focused
investigation, we examined the effects of mRNA COVID-19 vaccination on microRNA expression in
Citation: Shen, C.-J.; Lin, Y.-P.; Chen,
pregnant women. Key microRNAs, including miR-451a, miR-23a-3p, and miR-21-5p, were analyzed
W.-C.; Cheng, M.-H.; Hong, J.-J.; Hu,
for expression changes post-vaccination. Additionally, we assessed variations in S1RBD IgG levels
S.-Y.; Shen, C.-F.; Cheng, C.-M.
and specific cytokines to gauge the broader immunological response. Results: Post-vaccination,
COVID-19 Vaccination in Pregnancy:
Pilot Study of Plasma MicroRNAs
significant expression shifts in the targeted microRNAs were observed. Alongside these changes,
Associated with Inflammatory we noted alterations in S1RBD IgG and various cytokines, indicating an adapted inflammatory
Cytokines after COVID-19 mRNA response. Notably, these immunological markers displayed no direct correlation with S1RBD IgG
Vaccination. Vaccines 2024, 12, 658. concentrations, suggesting a complex interaction between the vaccine and the immune system
https://doi.org/10.3390/vaccines in pregnant women. Conclusions: Our pilot study provides valuable insights into the nuanced
12060658 effects of the mRNA COVID-19 vaccine on immune dynamics in pregnant women, particularly
Academic Editor: Ralph A. Tripp
emphasizing the role of microRNAs. The findings illuminate the intricate interplay between vaccines,
microRNAs, and immune responses, enhancing our understanding of these relationships in the
Received: 2 May 2024
context of pregnancy. This research contributes significantly to the growing body of knowledge
Revised: 11 June 2024
regarding mRNA COVID-19 vaccines and their specific impact on maternal immunology, offering a
Accepted: 11 June 2024
foundation for further studies in this vital area.
Published: 14 June 2024

Keywords: COVID-19 vaccine; mRNA vaccine; microRNA; S1RBD IgG; pregnancy; cytokine

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and In November 2019, there was an outbreak of pneumonia of unknown cause in Wuhan,
conditions of the Creative Commons China, and a new coronavirus “SARS-CoV-2” emerged [1]. In just over two months, the
Attribution (CC BY) license (https://
number of confirmed cases of “COVID-19” worldwide exceeded 10,000. This is a rapid
creativecommons.org/licenses/by/
spread of disease unprecedented in this century. As of May of 2024, the epidemic continues
4.0/).

Vaccines 2024, 12, 658. https://doi.org/10.3390/vaccines12060658 https://www.mdpi.com/journal/vaccines

Vaccines 2024, 12, 658 2 of 24

to undergo changes [2]. Although scientists have made efforts to accelerate research, the
characteristics and trajectory of the epidemic remain unpredictable and rife with variables. To
ameliorate the disastrous effects brought on by COVID-19, vaccination is the most effective
response. In response to the urgency of the epidemic and the needs of the world, the COVID-
19 vaccine was developed and released to the public following rigorous efficacy and safety
testing [3,4]. Effective vaccination strategies or approaches being necessary to arrest the
pandemic, some studies have noted that mRNA vaccines such as the Moderna (mRNA-1273)
and Pfizer-BioNTech BNT162b2 vaccines appear to be more effective among all the types of
COVID-19 vaccines currently available [5–9].
In special groups, vaccination strategy must be more carefully considered. Vaccination is
completely safe during pregnancy and can provide excellent protection for newborns [10–12].
Both the fetus and pregnant women are at risk of infection during pregnancy [13,14]. The
American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal–
Fetal Medicine (SMFM) recommend that pregnant or breastfeeding women get the COVID-
19 vaccine [15]. From 2019 to now, multiple studies have confirmed that administering the
COVID-19 vaccine during pregnancy does not lead to an increased risk of adverse perinatal
outcomes [16,17].
MicroRNAs can regulate gene expression in many organisms, affect the mechanism of
human cancer, and are highly valued molecules in modern medicine [18]. After binding
to the three-terminal untranslated region (3’UTR) [19] of the target mRNA, the regulation
of gene expression ensues, and protein translation is promoted or inhibited. There is some
evidence that some microRNAs are dysregulated in COVID-19 patients. One study identified
miR-146a-5p, miR-21-5p, and miR-142-3p as the potential biomarkers of COVID-19 severity
that could also be therapeutically targeted [20]. Another study indicated that miR-423-5p,
miR-23a-3p, and miR-195-5p could independently identify COVID-19 cases and distinguish
SARS-CoV-2 from influenza infection [21]. Many studies examine microRNAs in patients
with COVID-19, but little research is available on the regulation of microRNAs following
COVID-19 vaccination. Yusuke Miyashita et al. [22] identified several immunomodulatory
microRNAs, including miR-126, miR-132, miR-221, and miR-625-3p, that were associated with
partial immune responses to COVID-19 vaccination. Additionally, miR-126 and miR-451a
correlate with TNF-α levels after receiving the initial dose of the COVID-19 vaccine.
There are also many immune-related cytokines associated with the regulation of mi-
croRNAs. For example, miR-451a is an IL-6R translational repressor that exacerbates the
IL-6-induced cytokine storm [23] and one of the top five downregulated microRNA genes in
COVID-19 patients [24]. One study demonstrated that the presence of miR-451a significantly
suppressed TNF-α and significantly increased IL-10 [25]. Another scientific study showed that
miR-21-5p had an anti-inflammatory effect that could inhibit the expression of IL-1β [26,27].
To better understand immune response following the administration of mRNA vaccine and
the
Vaccines 2024, 12, relationship
x FOR PEER REVIEW between related cytokines and microRNA, we conducted 3 ofa24series of studies

on 111 pregnant women. Figure 1 shows the experimental design of our pilot study.

Figure 1. Illustrates the experimental design of our pilot study. Peripheral blood samples were collected
Figure 1. Illustrates the experimental design of our pilot study. Peripheral blood samples were col-
from pregnantlected
women who received
from pregnant women who mRNA
receivedCOVID-19
mRNA COVID-19 vaccines, and
vaccines, and these samples
these samples werewere used for mi-
used for microRNA analysis and ELISA. The microRNA analysis was performed to identify differ-
croRNA analysis and ELISA. The microRNA analysis was performed to identify
entially expressed microRNAs post-vaccination, while ELISA was used to measure the concentra- differentially expressed
tions of S1RBD IgG and
microRNAs post-vaccination, neutralizing
while ELISA antibodies againstto
was used wild-type
measure and the
different variants, and vari-
concentrations of S1RBD IgG and
ous cytokines, including IL-6, IL-6R, TNF-α, IL-10, and IL-1β (created using BioRender.com).
neutralizing antibodies against wild-type and different variants, and various cytokines, including IL-6,
IL-6R, TNF-α,2.IL-10,
Materials
andand Methods
IL-1β (created using BioRender.com, accessed on 9 June 2024).
2.1. Participants and Sample Collection
This prospective study was approved by the Institutional Review Board of
Kaohsiung Medical University Hospital (IRB No. KMUHIRB-SV(II)-20210087). The study
enrolled pregnant women aged 20 or above who had no history of preterm labor, chronic
illnesses requiring immunosuppressants, cancer requiring specific treatments, pregnancy-
related illnesses such as gestational hypertension or diabetes, or previous COVID-19 dis-
ease and SARS-CoV-2 infection. All the eligible participants provided written informed
consent before enrollment. We specifically selected the cases where the interval between
Vaccines 2024, 12, 658 3 of 24

2. Materials and Methods

2.1. Participants and Sample Collection
This prospective study was approved by the Institutional Review Board of Kaohsiung
Medical University Hospital (IRB No. KMUHIRB-SV(II)-20210087). The study enrolled
pregnant women aged 20 or above who had no history of preterm labor, chronic illnesses
requiring immunosuppressants, cancer requiring specific treatments, pregnancy-related
illnesses such as gestational hypertension or diabetes, or previous COVID-19 disease and
SARS-CoV-2 infection. All the eligible participants provided written informed consent
before enrollment. We specifically selected the cases where the interval between the
last dose of the vaccine and delivery was between 4 and 8 weeks because the antibody
concentration generated during this period was at its peak (plateau phase). This time
window ensured the highest antibody transfer efficiency to the fetus as well. In addition,
we collected and analyzed liver and kidney function tests and coagulation profiles for the
participants who underwent cesarean section. The coagulation function test was performed
for the participants who received painless labor during vaginal delivery. These biochemical
parameters were within normal ranges, meaning no significant adverse effects on the liver,
kidney, or coagulation function in our study population.
Peripheral blood samples were obtained for microRNA analysis from 13 mothers
on the day of delivery and plasma samples were obtained from 3 non-pregnant healthy
women. Additionally, peripheral blood samples were collected for ELISA analysis from
111 mothers on the day of delivery. Regarding the 111 patients from whom the ELISA
analysis samples were obtained, 54 completed three doses of the vaccine, 49 completed
two doses of the vaccine, and 8 completed one dose. Both the two-dose vaccine and the
one-dose vaccine recipients received the Moderna (mRNA-1273) vaccine. Among the
three-dose recipients, all received the Moderna vaccine as their third dose, whereas their
first and second doses were sets of either the Moderna, the Oxford/AstraZeneca, or the
Pfizer-BioNTech BNT162b2 vaccine. None of the pregnant demonstrated any symptoms
related to COVID-19 during pregnancy.
Combining all the vaccines received, the participants were classified into one of
three groups as follows: (1) the three-dose group, whose participants received either
three doses of COVID-19 vaccine with Tdap and flu vaccination (n = 23), three doses
of COVID-19 vaccine with Tdap vaccination (n = 24), or only three doses of COVID-19
vaccine (n = 7); (2) the two-dose group, whose participants received either two doses of
COVID-19 vaccine with Tdap and flu vaccination (n = 7), two doses of COVID-19 vaccine
with Tdap vaccination (n = 21), or only two doses of COVID-19 vaccine (n = 21); and (3) the
one-dose group, whose participants received either one dose of COVID-19 vaccine with
Tdap vaccination (n = 2), or only one dose of COVID-19 vaccine (n = 6).

2.2. microRNA qPCR Analysis

Total RNA was isolated from 200 µL of plasma with a miRNeasy Serum/Plasma
Advanced Kit (Cat. No. 217204, Qiagen, Hilden, Germany) following the manufacturer’s
protocol. The plasma RNA samples were eluted in 20 µL nuclease-free water. The concen-
tration of the extracted total RNA sample was quantified using a Thermo Fisher’s Qubit
microRNA Assay Kit (Q32880, Hsinchu, Taiwan).
We investigated the real-time microRNA expression profiles with a MIRAscan and
NextAmp™ Analysis System (designed by Inti Taiwan, Inc.; manufactured by Quark
Biosciences, Inc., Zhubei, Taiwan), which was pre-printed with microRNA-specific primers.
To identify differentially expressed microRNAs, the microRNA expression profiles were
first normalized by using the quantile normalization method. The resulting microRNA
expression profiles were normalized by the expression level of the qPCR spike-in control
using the following formula:

∆Cq value = 20 − (Cq value − control)

Vaccines 2024, 12, 658 4 of 24

The higher the resulting value, the higher the microRNA expression level. Any
microRNAs without amplification signals across all the profiles were removed. The differ-
entially expressed microRNAs were filtered by 2 criteria: delta Cq ≥ 0.585 or ≤−0.585 and
p-value < 0.05.
For microRNA enrichment analysis, we used the differentially expressed microRNAs
as input and analyzed microRNA target interaction (MTI) in miRTarBase [28]. miRTarBase
is one of the largest experimentally validated databases for microRNA target analysis. MTIs
with more than 1 strong evidence or more than 2 weaker evidence or paper reports were
retained. Finally, the filtered target gene list was used for the enrichment analysis using
the R package clusterprofiler [29] according to the pathways or gene functional groups
from Gene Ontology (GO), KEGG Pathway, Reactome pathway, WikiPathways, Disease
Ontology, and DisGeNET [30–34].

2.3. ELISA
The concentration of IL-6, IL-6R, TNF-α, IL-10, IL-1β, and human IgG antibody against
SARS-CoV-2 S1 RBD protein in plasma were analyzed from the samples taken on the day of
delivery using the R & D system Quantikine Human Immunoassay for IL-6, IL-6R, TNF-α,
IL-10, IL-1β, and RayBio® COVID-19 S1 RBD protein Human IgG ELISA Kit (RayBiotech,
Peachtree Corners, GA, USA).
The different variants of neutralizing antibodies, including the BA.1, BA.2, BA.4,
and BA.5 variants, were determined using the GenScript SARS-CoV-2 Surrogate Virus
Neutralization Test Kit for wild-type and AcroBiosystem Neutralizing Antibody Titer
Serologic Assay Kit. The results were used to calculate the neutralizing antibody inhibition
rate based on the following formula:
 
OD value in 450 nm of sample
Inhibition(%) = 1 − × 100%
average OD value in 450 nm of negative control

The experiment was stopped by adding a stop solution and the color change from
blue to yellow was evaluated. The OD value absorbance (color intensity) at 450 nm was
read using a microplate spectrophotometer (Molecular Devices, San Jose, CA, USA). All
the ELISA results were obtained per the manufacturer’s protocol.

2.4. Statistical Analysis

The data were analyzed using GraphPad Prism 8 (GraphPad Software, San Diego, CA,
USA) and R Project for Statistical Computing (R version 4.2.2, Auckland, New Zealand).
The p-value was analyzed by using the two sample Student’s t-test to compare the two
different vaccine combinations, i.e., one, two, or three doses of COVID-19 vaccine with or
without Tdap or influenza vaccine. The error bars represent the standard deviation (SD).
All the results with p < 0.05 were considered statistically significant. Principal component
analysis (PCA) was used to evaluate any differences among the samples and treatment
conditions. An orthogonal transformation was performed to convert a set of observa-
tions of possibly correlated variables into a set of values of uncorrelated variables called
principal components.

3. Results
3.1. Participant Characteristics
Tables 1 and 2 display the participant demographic and clinical characteristics. Table 1
displays the data for the 16 participants whose plasma samples were used for the microRNA
analysis. Table 2 displays the data for the 111 participants whose samples were used for
the ELISA analysis. These 111 participants were all vaccinated with the Moderna vaccine
as their third dose. The participants who received only one dose or two doses all received
Moderna vaccines. For the group of participants who received three doses, the first and
second doses of the vaccine were sets of either the Moderna, Oxford/AstraZeneca, or Pfizer-
BioNTech BNT162b2 vaccine. No participants demonstrated symptoms related to COVID-
Vaccines 2024, 12, 658 5 of 24

19 during pregnancy. The median maternal age in the one-dose group was 34.25 years (IRQ
38–28.5). The median maternal age in the two-dose group was 32.37 years (IRQ 35–28). The
median maternal age in the three-dose group was 33.57 years (IRQ 37.75–30). Among the
111 participants, 47 received pertussis vaccine, 30 received influenza and pertussis vaccine,
and 34 received neither influenza nor pertussis.

Table 1. Participant demographic and clinical data for microRNA analysis.

Clinical Data
Weeks of The Interval between the
Sample Age Gestation at Last Dose of the COVID-19 Kind of COVID-19 Vaccine for
Parity BMI
ID (Year) Delivery Vaccine and the Day of the First/Second/Third Dose
(Weeks) Delivery (Weeks)
Control 1 - 23 19.8 - - BNT/BNT/Moderna
Control 2 - 23 18.62 - - BNT/BNT/Moderna
Control 3 - 23 21 - - BNT/BNT/Moderna
No dose 1 3 40 28.38 39 - -
No dose 2 1 40 26.95 37 - -
No dose 3 4 38 30.12 36 - -
One dose 1 1 36 25 36 15 Moderna/-/-
One dose 2 2 25 26.8 37 6 Moderna/-/-
One dose 3 0 37 25 39 6 Moderna/-/-
Two dose 1 3 40 25.7 39 8 Moderna/Moderna/-
Two dose 2 0 31 25.71 38 5 Moderna/Moderna/-
Two dose 3 1 33 28 40 4 Moderna/Moderna/-
Three dose 1 2 35 21.8 40 6 Moderna/Moderna/Moderna
Three dose 2 1 24 27.6 38 5 Moderna/Moderna/Moderna
Three dose 3 1 32 23.82 40 5 Moderna/Moderna/Moderna
Three dose 4 2 35 23.52 39 6 Moderna/Moderna/Moderna

Table 2. Participant demographic and clinical data for ELISA.

Included in the Analysis

Variable a
One Dose Two Dose b Three Dose c
34.25 * ± 6.58 ** 32.49 ± 4.66 33.57 ± 5.36
Age of mothers (years)
IQR 38–28.5 IQR 35–28 IQR 37.75–30
1.375 * ± 0.74 ** 1.31 ± 0.55 1.57 ± 0.63
Parity
IQR 1.25–0 IQR 2–1 IQR 2–1
BMI 26.93 * ± 4.13 ** 27 ± 4.41 27.29 ± 3.99
(body mass index) IQR 28.19–23.68 IQR 30.3–23.82 IQR 29.61–24.3
Weeks of gestation at the first dose of COVID-19 33.5 ± 2.27 23.06 ± 10.26 0.74 ± 8.93
vaccination (weeks) IQR 35–32.5 IQR 28–24 IQR 4.75–(−5.57)
Weeks of gestation at the second dose of COVID-19 32.82 ± 1.91 14.48 ± 7.29
-
vaccination (weeks) IQR 34–31 IQR 20–9.5
Weeks of gestation at the third dose of COVID-19 33.33 ± 2.49
- -
vaccination (weeks) IQR 35–31
The interval between the first dose of COVID-19 vaccination
4.75 ± 1.91 15.27 ± 10.08 37.43 ± 9.05
and the collection of blood samples (the day of
IQR 6–3.75 IQR 15–10 IQR 42–34
delivery) (weeks)
Vaccines 2024, 12, 658 6 of 24

Table 2. Cont.

Included in the Analysis

Variable a
One Dose Two Dose b Three Dose c
The interval between the second dose of COVID-19 vaccination
5.67 ± 1.76 23.72 ± 7.39
and the collection of blood samples (the day of -
IQR 7–5 IQR 29–19
delivery) (weeks)
The interval between the third dose of COVID-19 vaccination 5.11 ± 2.03
- -
and the collection of blood samples (day of delivery) (weeks) IQR 7–4
38.25 ± 0.89 38.49 ± 1.17 38.44 ± 1.11
Weeks of gestation at delivery (weeks)
IQR 39–37.75 IQR 39–38 IQR 39–38
3123.13 * ± 363.52 ** 3089.9 ± 360.78 3103.89 ± 311.99
Weight of newborn (g)
IQR 3205–2992.5 IQR 3235–2900 IQR 3293.75–2883.8
Sex of newborn
Male 4 (50% ***) 22 (44.9% ***) 28 (51.9% ***)
Female 4 (50% ***) 27 (55.1% ***) 26 (48.1% ***)
* mean; ** standard deviation (± SD); *** percentage of all the surveyed patients. a = one dose, n = 8; b = two dose,
n = 49; c = three dose, n = 54; IQR: interquartile range.

3.2. The Different Expression Levels of microRNA

After the microRNA array analysis, we used the following formula to normalize
the generated microRNA expression profile through the expression level of the qPCR
spike-in control:
∆Cq value = 20 − (Cq value − control)
The higher the result value, the higher the expression level of microRNA; Figure 2
displays the microRNA values with differential expression from 16 samples. We used
principal component analysis (PCA), performing an orthogonal transformation to convert
the observed values of a set of possibly correlated variables into the values of a set of
uncorrelated variables. The difference is shown in Figure 3. The sample values that deviated
significantly from the values obtained from the samples of the subjects receiving the same
vaccine dose were excluded. After excluding the discrete samples, the eight microRNAs
shown at the bottom of Figure 2 were clearly distinguished during the grouping process.
These represented the microRNAs that are expressed in most samples and were used to
create a heatmap drawing with a total of eight microRNAs (Figure 4). Three microRNAs
exhibited the highest levels of expression, i.e., miR-451a, miR-23a-3p, and miR-21-5p.
We inputted the three microRNAs that showed significant differences in expression
levels and then performed a gene set enrichment analysis on the pathways and gene
ontology. Among the top five biological process enrichment GO terms shown in Tables 3–5,
several of them were found to be associated with inflammation or interleukin, suggesting
the potential involvement of these microRNAs in regulating inflammatory processes.

Table 3. Top 5 enriched terms for miR-451a.

miR-451a
Pathway Description Gene Name p-Value
Interleukin-4 and Interleuki-13 Signaling AKT1/MMP2/MMP9/BCL2/MYC/IL6R 2.60 × 10−7
Vascular Inflammations AKT1/MMP2/MMP9/ADAM10/CRP/ETS1/CAV1 4.09 × 10−7
Immune Thrombocytopenic Purpura MIF/ABCB1/MMP9/BCL2/IKBKB/ADAM10/CRP 9.75 × 10−7
Acquired Immunodeficiency Syndrome ABCB1/MMP2/MMP9/BCL2/MYC/CRP 2.31 × 10−6
Pelvic Inflammatory Disease MMP2/MMP9/CRP 1.32 × 10−5
Target Cytokine related to COVID-19 [23–25] IL-6R/IL-6/TNF-α [23–25]
 levels and then performed a gene set enrichment analysis on the pathways and g
tology. Among the top five biological process enrichment GO terms shown in Ta
several of them were found to be associated with inflammation or interleukin,
Vaccines 2024, 12, 658 7 of 24
su
the potential involvement of these microRNAs in regulating inflammatory proce

Figure 2. Heatmap of microRNA expression profiles from the 16 peripheral blood samples of the
Figure 2. Heatmap of microRNA expression profiles from the 16 peripheral blood samp
pregnant women who received varying doses of the mRNA COVID-19 vaccine and the non-pregnant
pregnant womencontrols.
who received
The heatmap varying
visualizes thedoses of the mRNA
relative expression COVID-19
levels of microRNAs, vaccine
with eight and the n
microRNAs
nant controls. The heatmap visualizes the relative expression levels of microRNAs, with
(highlighted with red frame lines) exhibiting distinct expression patterns among the sample groups.

croRNAs (highlighted with red frame lines) exhibiting distinct expression patterns among
ple groups.

Table 3. Top 5 enriched terms for miR-451a.

REVIEW 9 of

Vaccines 2024, 12, 658 8 of 24

Figure 3. PCA graph of the 16 samples (red circles on the graph are 4 outliers). All 16 samples
Figure 3. PCA graph ofwere
thedivided
16 samples (redbased
into five groups circles
on theon the graph
administered aredosage.
vaccine 4 outliers). All 16thesamples
After performing PCA w
divided into five groups based on the administered vaccine dosage. After performing the PCA an
analysis, we observed that four samples (No_dose1, One_dose1, Control2, and Three_dose4) were

ysis, we observed that distant

four from the other samples within their respective groups, indicating potential outliers that could
samples (No_dose1, One_dose1, Control2, and Three_dose4) were d
affect the analysis results. Therefore, these samples were excluded from subsequent analyses.
tant from the other samples within their respective groups, indicating potential outliers that co
affect the analysis results. Therefore, these samples were excluded from subsequent analyses.
PEER REVIEW
Vaccines 2024, 12, 658 9 of 24

Figure 4. FocusedFigure 4. Focused

heatmap of heatmap of the 8 microRNAs
the 8 microRNAs with significant
with significantexpression differences among
expression the
differences am
12 peripheral blood samples. The three microRNAs highlighted with red frame lines (miR-451a, miR-
12 peripheral blood samples.
23a-3p, Theshow
and miR-21-5p) three microRNAs
the most highlighted
pronounced expression with
changes and red frame
were selected lines (m
for further
miR-23a-3p, and miR-21-5p) show
investigation of their the roles
potential most pronounced
in the immune responseexpression changesvaccination
to the mRNA COVID-19 and were sel
during pregnancy.
further investigation of their potential roles in the immune response to the mRNA COVID
cination during pregnancy.

3.3. The Level of IL-6, IL-6R, TNF-α, IL-10, IL-1β, and S1RBD IgG in Samples from Pat
Receiving Different Doses of COVID-19 Vaccine
Vaccines 2024, 12, 658 10 of 24

Table 4. Top 5 enriched terms for miR-21-5p.

miR-21-5p
Pathway Description Gene Name p-Value
PTEN/FAS/EGFR/IL1B/ICAM1/PLAT/PTX3/
TNFAIP3/CCL20/DUSP10/PPARA/NTF3/FASLG/
Inflammation 2.79 × 10−11
SMAD7/MMP2/VEGFA/MMP9/STAT3/MYD88/
IL12A/CXCL10/CLU/CCR7/TLR2
BCL2/SOCS5/MYC/IL1B/ICAM1/FASLG/SOX2/
Interleukin-4 and Interleukin-13 Signaling 6.62 × 10−10
MMP2/VEGFA/MMP9/STAT3/BCL6/IL12A/FOXO3
BCL2/PTEN/FAS/MYC/IL1B/ICAM1/SP1/FASLG/
Acquired Immunodeficiency Syndrome 1.96 × 10−9
MMP2/VEGFA/MMP9/SLK/STAT3/BCL6/MYD88/CXCL10
RASGRP1/BCL2/JAG1/TIMP3/PTEN/FAS/IL1B/
Immune Thrombocytopenic Purpura TNFAIP3/FASLG/SMAD7/VEGFA/MMP9/STAT3/ 3.41 × 10−8
BCL6/IL12A/CXCL10/GP5
TIMP3/PTEN/PPIF/IL1B/ICAM1/PTX3/SMARCA4/
Vascular Inflammations SMAD7/MMP2/VEGFA/MMP9/AKT2/STAT3/ 4.90 × 10−8
FOXO3/CXCL10/TLR2
Target Cytokine related to COVID-19 [26,27] IL-1β/IL-12 [26,27]

Table 5. Top 5 enriched terms for miR-23a-3p.

miR-23a-3p
Pathway Description Gene Name p-Value
CXCL12/PTEN/CDH1/IRF1/FAS/MT2A/
Inflammation 2.30 × 10−5
STS/VCAM1/TNFAIP3/TLR6/FGF2/FOXP3/SIRT1
CXCL12/CDH1/IRF1/FAS/VCAM1/SOD2/
Human Immunodeficiency Virus Infectious Disease 2.96 × 10−5
MEF2C/FOXP3/BCL2/SIRT1
FOXO3/VCAM1/ADAM17/FGF2/
Experimental Autoimmune Encephalomyelitis 5.85 × 10−5
TGFB2/SIRT1
Autoimmune Lymphoproliferative Syndrome FAS/TNFAIP3/XIAP/FOXP3/BCL2 8.79 × 10−5
CXCL12/HES1/PTEN/FAS/HOXB4/STS/
Immune Thrombocytopenic Purpura 0.000127
TNFAIP3/FGF2/FOXP3/BCL2
Target Cytokine or Protein related to COVID-19 [24] IL-6/BCL2 [24]

3.3. The Level of IL-6, IL-6R, TNF-α, IL-10, IL-1β, and S1RBD IgG in Samples from Patients
Receiving Different Doses of COVID-19 Vaccine
We carried out two classification methods for the experimental data regarding the
quantification of IL-6, IL-6R, TNF-α, IL-10, IL-1β, and S1RBD IgG: (1) classification according
to the number of doses administered and (2) classification based on the number of weeks
between the third-dose vaccine administration and the time of delivery. Figure 5 displays
the trend in cytokine and IgG concentrations. The concentration of both in the samples from
the groups receiving three doses of mRNA vaccines was higher than those who received two
doses or one dose, and there were also significant differences between the groups. The three-
dose group was used for classification. The concentration of IgG antibodies has a downward
trend with the number of weeks, and the changes in other cytokines are quite different. From
the perspective of IL-6, IL-6R, and IL-1β, the concentration at 7–8 weeks is the lowest. This
may be because the time interval from vaccination was longer. Notably, the same results exist
for all the cytokines, with the highest concentration at 5–6 weeks. TNF-α appears to be the
primary inflammatory factor compared with the other factors. The number of weeks does not
seem to have a significant impact on concentration. The concentration of IL-10, a cytokine of
anti-inflammatory response, was higher at 7–8 weeks than at 3–4 weeks.
Vaccines 2024, 12, x FOR PEER REVIEW 11 of 24

Vaccines 2024, 12, 658 concentration. The concentration of IL-10, a cytokine of anti-inflammatory response, was
11 of 24
higher at 7–8 weeks than at 3–4 weeks.

Figure 5. Cont.
Vaccines 2024, 12, x FOR PEER REVIEW 12 of 24
Vaccines 2024, 12, 658 12 of 24

(a)S1RBD
Figure5.5.(a)
Figure S1RBDIgG,
IgG,(b)
(b) IL-6,
IL-6, (c) IL-6R,
IL-6R, (d)
(d)TNF-α,
TNF-α,(e) (e)IL-10,
IL-10,and (f)(f)
and IL-1β
IL-1βconcentrations.
concentrations.TheThe
figures
figuresononthe
theleft
leftshow
showthe
thesamples
samples classified accordingtotothe
classified according thenumber
numberofof injections.
injections. TheThe figures
figures on on
the
theright
rightshow
showthe thesamples
samples classified
classified by the the number
numberof ofweeks
weeksbetween
betweenthe thethird
third dose
dose of of injection
injection
time
timeand
andthe
theday
dayofofdelivery.
delivery. ** p << 0.05,
0.05, ** p < 0.01,
0.01, ***
*** pp<< 0.001,
0.001,****
****pp<<0.0001.
0.0001.

WeWeexamined
examinedwhether
whetherthe the administrationofof
administration pertussis
pertussis and
and influenza
influenza vaccination
vaccination pro-
produced changes in cytokine concentration. Figure 6 displays the concentration
duced changes in cytokine concentration. Figure 6 displays the concentration of each cy- of each
cytokine
tokine for for
thethe
two-two-andand three-dosegroups.
three-dose groups. The
The cytokine
cytokine concentration
concentrationfor forthe samples
the samples
from the one-dose group is presented in Supplementary Figure S1. The
from the one-dose group is presented in Supplementary Figure S1. The administration administration of of
the pertussis vaccine, pertussis plus influenza vaccine, and the type of vaccine used for
the pertussis vaccine, pertussis plus influenza vaccine, and the type of vaccine used for
the first and second doses (AZ or BNT) do not appear to significantly impact the cytokine
the first and second doses (AZ or BNT) do not appear to significantly impact the cytokine
concentration. The statistical analysis shows no significant difference (p = ns). While
concentration. The statistical analysis shows no significant difference (p = ns). While the
the initial comparison was limited to those groups receiving one, two, or three vaccine
initial
doses,comparison
there were was also limited
variationsto those groups
observed receiving
within one, two,
these groups, or three vaccine
depending doses,
on whether
there were also variations observed within these groups, depending on whether
pertussis was present or not. These differences were observed to varying degrees. These pertussis
was present
findings or not.
indicate These
that we candifferences
exclude thewere observed
pertussis to varying
vaccine degrees.
and influenza These
vaccine findings
variables.
indicate that we
Additionally, thecan
typeexclude
of first the
andpertussis vaccine
second doses and to
appears influenza
have no vaccine
effect onvariables. Addi-
the need for a
tionally,
booster the
dosetype
of theofModerna
first andvaccine.
second doses appears to have no effect on the need for a
booster dose of the Moderna vaccine.
Vaccines 2024, 12, x FOR PEER REVIEW 13 of 24
Vaccines 2024, 12, 658 13 of 24

Figure
Figure6. 6.IL-6,
IL-6,IL-6R,
IL-6R,TNF-α,
TNF-α,IL-10,
IL-10, and
and IL-1β concentrations
concentrations(week (weekrange
range= =1–81–8weeks;
weeks;twotwo doses
doses
with
withpertussis
pertussisvaccine,
vaccine,nn==21,
21,without
without pertussis vaccine, nn== 21,
pertussis vaccine, 21,and
andwith
withpertussis
pertussis and
and influenza
influenza
vaccines,
vaccines,n n= =7;7;three
threedoses
doses with AZand
with AZ andpertussis
pertussis vaccines,
vaccines, n =n13,
= 13,
onlyonly
withwith pertussis
pertussis vaccine,
vaccine, n = 11,n =
11,without
without pertussis vaccine, n = 7, and with pertussis and influenza vaccines, n = 23.
pertussis vaccine, n = 7, and with pertussis and influenza vaccines, n = 23. Correlation of allCorrelation
of cytokines
all cytokinesand andS1RBDS1RBD
IgG. IgG.
ns, p ns, p > 0.05,
> 0.05, * p < **
* p < 0.05, 0.05,
p < ** p <***
0.01, 0.01,
p <*** p < 0.001,
0.001, **** p <**** p < 0.0001.
0.0001.

To determine whether there was any correlation between the five cytokines and
whether they influence each other, we used the R program software (version 4.2.2) to
Vaccines 2024, 12, 658 14 of 24

To determine whether there was any correlation between the five cytokines and
whether they influence each other, we used the R program software (version 4.2.2) to
conduct a correlation analysis among the five cytokines. Figure 7 displays the correlation
between all the cytokines. Except in the cases of SARS-CoV-2, which creates a cytokine
storm, the immune system provides an appropriate response, mediates resistance to in-
vading microorganisms, and enables host survival after infection in general. As the body
responds to vaccines, the persistently circulating low levels of pro-inflammatory cytokines
may be a natural consequence of innate and adaptive responses to the vaccination, as
compared to those diagnosed with COVID-19. After the third or even fourth dose of
the COVID-19 vaccine, the body maintains a low level of inflammatory cytokines. We
examined the correlation between the neutralizing antibodies and cytokines produced after
vaccination but found little to no relationship between the two (Supplementary Figure
S2). For different cytokines, we also drew an ROC curve to determine whether or not the
number of weeks could be used to classify cytokine concentration based on the AUC value.
An AUC of at least 0.8 indicates that it can be used as a good evaluation criterion. The
comparative graphs and results are provided in Figure 8 and Table 6.

Table 6. AUC of S1RBD IgG, IL-6, IL-6R, TNF-α, IL-10, and IL-1β over time.

0–8 Week 0–2 Week 3–4 Week 5–6 Week 7–8 Week
S1RBD IgG 0.8086 0.75 0.9394 0.9158 0.6875
IL-6 0.8887 1 0.9697 0.9323 0.6302
IL-6R 0.8107 0.8125 0.7273 0.8421 0.898
TNF-α 0.5911 0.5938 0.6717 0.6648 0.5362
IL-10 0.5865 0.7143 0.7143 0.6545 0.5357
IL-1β 0.7146 0.8571 0.899 0.7288 0.6039
Vaccines 2024, 12, x FOR PEER REVIEW 15 of 24

Vaccines 2024, 12, 658 15 of 24

Figure 7. Cont.
Vaccines 2024, 12, x FOR PEER REVIEW 16 of 24
Vaccines 2024, 12, 658 16 of 24

Figure 7. 7.The
Figure Thecorrelation
correlation analysis betweenthe
analysis between thefive
fivecytokines
cytokines (IL-6,
(IL-6, IL-6R,
IL-6R, TNF-α,
TNF-α, IL-10,
IL-10, and IL-1β)
and IL-1β) and S1RBD
and S1RBD IgG.
IgG. The The analysis
analysis was performed
was performed using the Rusing the R
software
software to determine
to determine whether whether thereany
there were were any significant
significant relationships
relationships among among these immune
these immune markers.markers. The Spearman
The Spearman’s s rank correlation
rank correlation coefficients
coefficients (R) and (R) and prob-
probability
ability density
density values
values (p)provided
(p) are are provided forpairwise
for each each pairwise comparison.
comparison. ** p <***
** p < 0.01, 0.01,
p < *** p < ****
0.001, 0.001,
p <**** p < 0.0001.
0.0001.
Vaccines 2024, 12, 658 17 of 24
Figure 8. ROC curves of S1RBD IgG, IL-6, IL-6R, TNF-α, IL-10 and IL-1β.

0–8 week
(a1) IgG (b1) IL-6 (c1) IL-6R (d1) TNF-α (e1) IL-10 (f1) IL-1β

0–2 week
(a2) IgG (b2) IL-6 (c2) IL-6R (d2) TNF-α (e2) IL-10 (f2) IL-1β

3–4 week
(a3) IgG (b3) IL-6 (c3) IL-6R (d3) TNF-α (e3) IL-10 (f3) IL-1β

Figure 8. Cont.
Vaccines 2024, 12, 658 18 of 24

5–6 week
(a4) IgG (b4) IL-6 (c4) IL-6R (d4) TNF-α (e4) IL-10 (f4) IL-1β

7–8 week
(a5) IgG (b5) IL-6 (c5) IL-6R (d5) TNF-α (e5) IL-10 (f5) IL-1β

Figure 8. The receiver operating characteristic (ROC) curves for S1RBD IgG and the five examined cytokines (IL-6, IL-6R, TNF-α, IL-10, and IL-1β) over different
time intervals post-vaccination. The ROC curves were generated to evaluate the ability of these immune markers to discriminate between different time points
after vaccination. The area under the curve (AUC) values were calculated for each marker and time interval, with an AUC of at least 0.8 indicating good
discriminatory power.
Vaccines 2024, 12, 658 19 of 24

4. Discussion
Maternal immunization is a universally recognized concept, believed to play a piv-
otal role in reducing the susceptibility of newborns to pathogenic infections. The post-
vaccination immune response, however, is influenced by a myriad of factors, including
age, gender, and immune status [35–38]. Pregnancy, a unique physiological state, leads
to notable shifts in steroid hormone levels, impacting immune cell functionality [39–41].
While there have been studies comparing the vaccine responses between pregnant and
non-pregnant individuals [42,43], there remains a paucity of research on such responses in
regard to SARS-CoV-2 vaccination.
Previous research indicates that microRNA is instrumental in modulating the sig-
naling pathways of the immune system [44–46]. However, the role of microRNA in post-
vaccination immune modulation, particularly in regard to the effects of mRNA vaccines on
inflammatory cytokine profiles, warrants further investigation. Our study endeavors to
elucidate the effects of COVID-19 vaccine and vaccine regimens on the immune regulatory
system of pregnant women by examining microRNA expression patterns. To compile
a comprehensive microRNA expression profile post-COVID-19 vaccination, we selected
samples from 3 vaccinated non-pregnant individuals and 13 pregnant individuals. After
excluding four outliers, we analyzed a total of 12 samples. Our findings reveal an upregu-
lation in the expression of eight microRNAs. Of these, three microRNAs, namely miR-451a,
miR-23a-3p, and miR-21-5p, displayed the most pronounced expression changes. We thus
chose these three candidate microRNAs for the in-depth regulation function analysis to
further delve into the immune response post-vaccination.
Our pilot study found that all the pregnant women who received the mRNA COVID-
19 vaccine experienced only mild local reactions at the injection site, such as pain, redness,
or swelling. Notably, no participants reported systemic side effects that required medical
attention or reporting to the adverse event monitoring system. The absence of significant
side effects in our study population is crucial, as it suggests that the immune response
observed in our investigation is not confounded by adverse reactions to the vaccine. This
finding underscores the importance of our results, as it demonstrates that the mRNA
COVID-19 vaccine is well tolerated in pregnant women and that the immune response we
characterized is likely a direct result of the vaccine itself rather than a consequence of any
side effects.
In our investigation, we discerned that the levels of S1RBD IgG, along with cytokines
IL-6, IL-6R, TNF-α, IL-10, and IL-1β, were markedly elevated in the individuals who re-
ceived two or three doses of the mRNA COVID-19 vaccine compared to those who received
a single dose. This observation underscores the efficacy of booster vaccinations in induc-
ing robust protective cytokine responses post-mRNA COVID-19 vaccination. While the
concentration of S1RBD IgG wanes over time post-vaccination, we noted that the cytokine
levels peaked between 5 and 6 weeks after vaccination. Interestingly, no correlation was
observed between the concentrations of S1RBD IgG and cytokine levels. This distinction
sheds light on the divergent roles of B cells and T cells post-vaccination. While B cells
generate neutralizing antibodies that thwart viral entry into cells, T cell immunity is pivotal
in halting disease progression and safeguarding against emergent viral variants.
The previous literature has emphasized the cardinal role of IL-6 in both innate and
adaptive immune regulation, with IL-6 known to activate B cells into antibody-secreting
plasma cells [47–49]. However, our study did not identify any correlation between antibody
concentrations and the levels of IL-6 or IL-6R. Furthermore, cytokines such as IL-6, TNF-
α, IL-10, and IL-1β have been previously linked to adverse effects post-vaccination and
infections. Research on mycoplasma pneumoniae membrane lipoprotein vaccine has
demonstrated that lipoprotein-induced vaccine-enhanced disease in a mouse model is
concomitant with elevated inflammatory cytokines in lung lavage fluid [50]. The elevated
serum levels of IL-6, IL-10, and TNF-α have been correlated with disease severity and
mortality in COVID-19 patients [51–53]. The cytokine triad of IL-1β, IL-6, and TNF is
associated with the post-acute sequelae of COVID-19 [54].
Vaccines 2024, 12, 658 20 of 24

Based on our research, the mRNA COVID-19 vaccine has been found to induce the
upregulation of three specific microRNAs: miR-146a-5p, miR-21-5p, and miR-142-3p. These
microRNAs are associated with the production of cytokines IL-6, IL-10, and TNF-α, which
are known to be generated during the natural immune response to SARS-CoV-2 virus
infection in humans. This suggests that the mRNA COVID-19 vaccine stimulates innate
immunity in a similar manner to a natural infection with the SARS-CoV-2 virus. Given the
potential implications of these cytokines in immune response, it is imperative that further
research be undertaken to comprehensively understand their significance post-vaccination.
While prior research has indicated that heterologous boosting can stimulate a more potent
antibody response compared to homologous boosting, our study did not observe any
influence of heterologous boosting on the cytokine concentrations under investigation.
Additionally, the administration of pertussis and influenza vaccines during pregnancy did
not exhibit any discernible impact on cytokine levels.
The main limitation of our pilot study is the inequality in sample sizes among the
vaccination groups, particularly the small number of participants in the single-dose group.
Due to the high vaccination rate among the pregnant women and the rarity of individuals
receiving only one dose in the current climate, it was extremely challenging to achieve
balanced group sizes despite our best efforts to maintain the integrity of our research data.
The smaller sample size in the single-dose group may have limited the statistical power to
detect significant differences compared to the other groups. While we attempted to control
for potential confounding factors by maintaining a consistent interval between the last
dose of vaccination and delivery (2–8 weeks) based on our previous findings, the timing of
vaccination during pregnancy varied among the participants. This variation could have
influenced the observed immune responses. Future studies should aim for larger sample
sizes and more balanced group distributions to further validate our findings. However,
this may prove difficult given the current challenges in recruiting single-dose individuals.
Researchers should also strive for tighter control over the timing of vaccination during
pregnancy to minimize potential confounding effects.
The second limitation of our pilot study is the lack of a direct comparison group consisting
of pregnant women who developed natural immunity after COVID-19 exposure. This prevents
us from quantitatively comparing the extent of the microRNA and cytokine increases between
the vaccinated individuals and those with natural immunity. Future research should aim
to include a cohort of pregnant women who have recovered from COVID-19 and compare
their microRNA and cytokine profiles to those of vaccinated pregnant women. This would
provide valuable insights into the quantitative differences in the immune response generated
by natural infection versus vaccination in this specific population.
The third study limitation is the heterogeneity in the primary vaccination series among
the three-dose recipients, who received either the Moderna, Oxford/AstraZeneca, or Pfizer-
BioNTech BNT162b2 vaccines for their first two doses. Although this variation could
potentially influence immune response, the evidence suggests that the type of primary
series may not significantly impact the effectiveness of the Moderna booster dose [55].
As all the three-dose recipients in our study received the Moderna vaccine as their final
dose, we believe that the impact of the heterogeneous primary series on our findings is
likely to be minimal. Furthermore, our study focused primarily on comparing the immune
responses between the dosage groups rather than directly comparing the vaccine types,
and by ensuring that all the participants within each dosage group received the same
vaccine for their last dose, we minimized the potential confounding effects of vaccine type
on our results.
Lastly, our pilot study lacks comprehensive control data from non-pregnant healthy
women. While our research focused on the immunological profiles of pregnant women
post-COVID-19 mRNA vaccination, comparing these findings with those of non-pregnant
individuals would have provided deeper insights into any potential differences in vaccine-
induced immune responses. Our study included a small control group of three non-
pregnant healthy women, which was insufficient for a robust comparative analysis. Future
Vaccines 2024, 12, 658 21 of 24

studies should include a larger cohort of non-pregnant healthy women to enhance the
statistical power and comprehensively compare the immune responses between pregnant
and non-pregnant individuals. This will help clarify how pregnancy-related immunological
changes might influence vaccine efficacy and immune regulation.
Our research modestly attempts to shed light on the immune response induced by
mRNA COVID-19 vaccination. Through our observations, we have noted the importance
of cytokines and have begun to explore the potential nuances of microRNA expression
patterns. We hope our findings contribute to the broader understanding of this topic.

5. Conclusions
Our analysis reveals the significant impact of the mRNA COVID-19 vaccine on the
immune regulatory system in pregnant women, particularly concerning specific microRNA
expression patterns. Notably, miR-451a, miR-23a-3p, and miR-21-5p showed increased
expression post-vaccination, suggesting their potential role in immune system signaling.
We also identified changes in S1RBD IgG and cytokine (IL-6, IL-6R, TNF-α, IL-10, and IL-1β)
levels in the vaccinated individuals, pointing to an augmented inflammatory response
in boosting individuals. Importantly, these immune markers were not correlated with
S1RBD IgG concentrations. In essence, the mRNA COVID-19 vaccine essentially influences
the immune response in pregnant women, with specific microRNAs playing a pivotal
role. These insights set the stage for deeper studies into the intricate relationship between
vaccines, microRNAs, and the immune system, especially during pregnancy.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/vaccines12060658/s1, Figure S1: IL-6, IL-6R, TNF-α, IL-10 &
IL-1β concentration (Week range = 1–8 weeks; one dose: w/Pertussis vaccine, n = 21; w/o Pertussis
vaccine, n = 21); Figure S2: Correlation between the neutralizing antibody inhibition rate of different
variants and IL-6, IL-6R, TNF-α, IL-10, IL-1β concentration and IL-6/IL-6R in pregnant women who
received three doses of mRNA vaccine (n = 42).
Author Contributions: Conceptualization, C.-J.S., C.-F.S., and C.-M.C.; methodology, Y.-P.L., W.-C.C.,
M.-H.C., J.-J.H., and S.-Y.H.; software, Y.-P.L., W.-C.C., M.-H.C., J.-J.H., and S.-Y.H.; validation, C.-
J.S., Y.-P.L., W.-C.C., M.-H.C., J.-J.H., and S.-Y.H.; formal analysis, C.-J.S., Y.-P.L., W.-C.C., M.-H.C.,
J.-J.H., and S.-Y.H.; investigation, C.-J.S., Y.-P.L., W.-C.C., M.-H.C., J.-J.H., S.-Y.H., C.-F.S., and C.-M.C.;
resources, C.-J.S., C.-F.S., and C.-M.C.; data curation, C.-J.S., Y.-P.L., W.-C.C., M.-H.C., J.-J.H., and
S.-Y.H.; writing—original draft preparation, C.-J.S., Y.-P.L., and C.-M.C.; writing—review and editing,
C.-J.S., C.-F.S., and C.-M.C.; visualization, Y.-P.L., W.-C.C., J.-J.H., and S.-Y.H.; supervision, C.-J.S.,
C.-F.S., and C.-M.C.; project administration, C.-J.S., C.-F.S., and C.-M.C.; funding acquisition, C.-M.C.
and C.-J.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Taiwan’s Kaohsiung Medical University Hospital (KMUH111-
1M33 and KMUH112-2M32), Taiwan’s National Tsing Hua University (113Q2302E1), and Taiwan’s
National Science and Technology Council (112-2622-E-007-028).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board of Kaohsiung Medical Uni-
versity Hospital (IRB No. KMUHIRB-SV(II)-20210087, an ethics review committee, on 7 August 2021).
Informed Consent Statement: Informed consent was obtained from all the subjects involved in the study.
Data Availability Statement: The data presented in this study are available in this article and
Supplementary Materials.
Conflicts of Interest: Authors Mei-Hsiu Cheng and Jun-Jie Hong were employed by the company
Inti Taiwan, Inc. The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential conflict of interest.
Vaccines 2024, 12, 658 22 of 24

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