Article

Enhancement of Vaccine-Induced T-Cell Responses by PD-L1
Blockade in Calves
Tomohiro Okagawa 1 , Satoru Konnai 1,2,3, *, Hayato Nakamura 2 , Otgontuya Ganbaatar 2 , Yamato Sajiki 2 ,Kei Watari 2 , Haruka Noda 4 , Mitsuru Honma 4 , Yukinari Kato 5,6 , Yasuhiko Suzuki 1,3,7,8 , Naoya Maekawa 1 ,Shiro Murata 1,2 and Kazuhiko Ohashi 1,2,9
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Citation: Okagawa, T.; Konnai, S.;
Nakamura, H.; Ganbaatar, O.; Sajiki,Y.; Watari, K.; Noda, H.; Honma, M.;
Kato, Y.; Suzuki, Y.; et al.
Enhancement of Vaccine-Induced
T-Cell Responses by PD-L1 Blockadein Calves. Vaccines 2023, 11, 559.
https://doi.org/10.3390/
vaccines11030559
Academic Editor: Hani Boshra
Received: 5 February 2023
Revised: 24 February 2023
Accepted: 25 February 2023

Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan
Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo 060-0818, Japan
Institute for Vaccine Research and Development (HU-IVReD), Hokkaido University, Sapporo 001-0021, Japan
Hokkaido Research Station, Snow Brand Seed Co., Ltd., Naganuma 069-1464, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan
Division of Bioresources, International Institute for Zoonosis Control, Hokkaido University,
Sapporo 001-0020, Japan
Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE),
Hokkaido University, Sapporo 001-0020, Japan
International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Correspondence: konnai@vetmed.hokudai.ac.jp

Abstract: Interactions between programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) cause functional
exhaustion of T cells by inducing inhibitory signals, thereby attenuating effector functions of T
cells. We have developed an anti-bovine PD-L1 blocking antibody (Ab) and have demonstrated
that blockade of the interaction between PD-1 and PD-L1 reactivates T-cell responses in cattle. In
the present study, we examined the potential utility of PD-1/PD-L1-targeted immunotherapy inenhancing T-cell responses to vaccination. Calves were inoculated with a hexavalent live-attenuatedviral vaccine against bovine respiratory infections in combination with treatment with an anti-PD-L1
Ab. The expression kinetics of PD-1 in T cells and T-cell responses to viral antigens were measured
before and after vaccination to evaluate the adjuvant effect of anti-PD-L1 Ab. PD-1 expression was
upregulated in vaccinated calves after the administration of a booster vaccination. The activation
status of CD4+ , CD8+ , and γδTCR+ T cells was enhanced by the combination of vaccination and PDL1 blockade. In addition, IFN-γ responses to viral antigens were increased following combinatorial
vaccination with PD-L1 blockade. In conclusion, the blockade of the PD-1/PD-L1 interaction enhances
T-cell responses induced by vaccination in cattle, indicating the potential utility of anti-PD-L1 Ab in
improving the efficacy of current vaccination programs.
Keywords: PD-L1; PD-1; T cell; live attenuated vaccine; immunotherapy; cattle

Published: 1 March 2023

1. Introduction
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/).

Vaccines are central to the control strategy for infectious diseases by preventing illness
and death in individuals and the spread of disease through populations. In veterinary
medicine, a variety of vaccines have been developed and used for the control of infectious
diseases in animals. The prevention of infection by vaccination is particularly important forlivestock raised in groups. In calves, whose immune systems are immature and naive tovarious pathogens, immunization with vaccines is essential and effective vaccine programs
are required to protect calves against infectious diseases. Various adjuvants are currently

Vaccines 2023, 11, 559. https://doi.org/10.3390/vaccines11030559

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

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added to vaccine products to enhance their efficacy. Live attenuated vaccines are the mostwidely used vaccines for livestock and can induce both cellular and humoral immunity [1].
Programmed death 1 (PD-1) is an immune checkpoint molecule predominantly expressed by T cells [2]. The binding of PD-1 to its ligand programmed death-ligand 1 (PD-L1),
which is expressed by antigen-presenting cells and immune cells, inhibits T-cell receptor(TCR) signaling and reduces the effector function of T cells in a process known as T-cell exhaustion [3,4]. Previous studies in cattle have reported that PD-1 expression is upregulated
in T cells and PD-L1 expression is upregulated in antigen-presenting cells and B cells during
chronic infections, thereby decreasing T-cell responsiveness to antigens and Th1 cytokine
responses [5–8]. Conversely, the effector function of T cells can be reactivated by inhibiting
PD-1/PD-L1 binding using anti-PD-L1 antibody (Ab) [9,10]. We previously developed
an anti-bovine PD-L1 rat-bovine chimeric Ab (chAb) which is able to block bovine PD1/PD-L1 binding [10]. In clinical trials using cattle, treatment with anti-bovine PD-L1 chAb
activated T-cell responses, including cell proliferation and Th1 cytokine production [10–12].
Thus, anti-bovine PD-L1 chAb is expected to have potential as a molecular-targeted drug
for inducing immune responses by preventing T-cell exhaustion in cattle.
T cells play an important role in the induction of immune memory following vaccination with attenuated live viruses [1]. However, the PD-1/PD-L1 pathway is posited toinhibit the induction of memory T cells responsible for cellular immunity in response tovaccines or infection [13]. A previous study in a mouse model of lymphocytic choriomeningitis virus (LCMV) infection reported that the blockade of PD-1/PD-L1 inhibitory signals
in exhausted CD8+ T cells in combination with therapeutic vaccination against LCMV
synergistically enhanced the functional responses of CD8+ T cells and decreased viral titers
in mice chronically infected with LCMV [14]. Furthermore, PD-1 inhibition by anti-PD-1 Abor soluble PD-1 protein has been shown to enhance vaccine-induced primary and memory
CD8+ T-cell responses, thereby producing an adjuvant effect via a novel mechanism ofaction, in mouse and rhesus macaque models [15,16]. However, studies on the adjuvant
effect of PD-1/PD-L1 inhibition have been limited to mouse and monkey experimental
models, with no studies to date examining the combinatorial efficacy of anti-PD-L1 oranti-PD-1 Abs with vaccines against viral infection in clinical settings.
Accordingly, the present study aimed to evaluate the adjuvant effect of a PD-1/PD-L1
inhibitor as an adjuvant to vaccination in cattle using a currently used commercial vaccine
and anti-bovine PD-L1 chAb. Two doses of a hexavalent vaccine against viruses causing
bovine respiratory infections were administered to calves in combination with anti-PD-L1
chAb. Changes in PD-1 expression by T cells, numbers of viral antigen-responding T cells,
and cytokine response were measured before and after vaccination.
2. Materials and Methods
2.1. Animals, Vaccination, and Sample Collection
The design of the animal experiment using calves is shown in Figure 1. Twelve malecalves of the Holstein breed ranging from two to three months of age were used in thepresent study. All calves received two intramuscular administrations of “KYOTOBIKEN”
Calfwin 6 Combo Live Vaccine (Kyoto Biken Laboratories, Uji, Japan), a hexavalent liveattenuated vaccine which consists of bovine respiratory syncytial virus (BRSV, rs-52 strain,>105.0 TCID50 ), bovine viral diarrhea virus (BVDV) type 1a (No.1255 strain, >103.0 TCID50 ),BVDV-2 (KZ1254 strain, >103.0 TCID50 ), bovine herpesvirus-1 (BHV-1, No.758-43 strain,>104.0 TCID50 ), parainfluenza virus type 3 (PIV3, BN-CE strain, >105.0 TCID50 ), and adenovirus type 7 (TS-GT strain, >103.0 TCID50 ) with a six-week interval. Six of the vaccinatedanimals were intravenously administered 2 mg/kg of anti-bovine PD-L1 rat-bovine chAb
(Boch4G12) [10] twice just after the primary and booster vaccinations. Peripheral blood
samples were collected from all calves at least once every two weeks following the primary
vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation using Percoll (GE Healthcare, Chicago, IL, USA),
washed three times with phosphate-buffered saline (PBS), and suspended in PBS. FreshVaccines 2023, 11, 559

adenovirus type 7 (TS-GT strain, >103.0 TCID50) with a six-week interval. Six of the vaccinated animals were intravenously administered 2 mg/kg of anti-bovine PD-L1 rat-bovine chAb (Boch4G12) [10] twice just after the primary and booster vaccinations. Peripheral blood samples were collected from all calves at least once every two weeks following
the primary vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated from
3 of 12
blood samples by density gradient centrifugation using Percoll (GE Healthcare, Chicago,
IL, USA), washed three times with phosphate-buffered saline (PBS), and suspended in
PBS. Fresh PBMCs or PBMCs cryopreserved in heat-inactivated fetal bovine serum (FBS;
PBMCs
PBMCs
cryopreservedin heat-inactivatedfetal bovine(FBS;sulfoxide
ThermoThermo orFisher
Scientific,
Waltham,
MA, USA) containing
10%serum
dimethyl
FisherScientific,
MA, USA)
10% dimethyl
sulfoxideAll(NacalaiTesque,(NacalaiTesque,Waltham,
Kyoto, Japan)
were containing
used in subsequent
experiments.
experimental
Kyoto,
Japan)
wereused in subsequent
experiments.
All experimental
were
procedures
wereconducted
with the approvalof the Ethics
Committeeproceduresof the Facultyof
conducted
withtheapprovaloftheEthicsCommitteeoftheFacultyof
Veterinary
Medicine,
Veterinary Medicine, Hokkaido University (approval #17-0024).
Hokkaido University (approval #17-0024).

FigureFigure 1.
1. Outline
Outline ofof aa pilot
pilot study
study ofof thethe combined
combined vaccination
vaccination with
with PD-L1
PD-L1 blockade.
blockade. Twelve
Twelve calves
calves
were
atat
days
0
were administrated
administratedintramuscularly
intramuscularlywith
with“KYOTOBIKEN”“KYOTOBIKEN”Calfwin
Calfwin6 6Combo
ComboLive
LiveVaccine
Vaccinedays
0 andof theanimals
were
intravenously
administered
2 mg/kgof anti-PD-L1
Abafterjustand42.42.
SixSixof theanimals
werealsoalso
intravenously
administered
2 mg/kgof anti-PD-L1
Ab justaftervaccinations
the vaccinations
at 0daysand
42. Peripheral
samplescollected
all calvesattheat daysand042.
Peripheral
bloodblood
samples
were werecollected
from fromall calvesat least
least
once
every
two
weeks.
once every two weeks.

2.2. Flow Cytometric Analysis of PD-1 Expression
Expression
Six-color analysis of PD-1-expressing
PD-1-expressing T cells
cells was
was performed
performed using
using cryopreserved
cryopreserved
PBMCs
PBMCs obtained
obtained fromfrom animals
animals before
before andand after
after booster
booster vaccination.
vaccination. PBMCs
PBMCs were
were thawed
thawed◦ C forandand incubatedincubated in
in PBSPBS containing
containing 10%
10% goat
goat serum
serum (Thermo(Thermo Fisher
Fisher Scientific)
Scientific) at
at 25
25 °C
for
15
min
to
prevent
nonspecificreactions.
Cells
were
then
washedandincubatedwith
anti15 min to prevent nonspecific reactions. Cells were then washed and incubated with antibovine
bovine PD-1
PD-1 monoclonalmonoclonal antibody
antibody (1D10F1,(1D10F1, rabbit
rabbit IgG,
IgG, in-house)
in-house) oror rabbit
rabbit IgG
IgG isotype
isotypecontrol(EPR25A,
Abcam,
Cambridge,
UK)
in
the
presenceofanti-TCR1-N24
mAb(GB21A,
control (EPR25A, Abcam, Cambridge, UK) in the presence of anti-TCR1-N24
mAb
mouse
IgG
State University
Monoclonal
AntibodyAntibody
Center, Pullman,WA,
2b ; Washington(GB21A,mouse
IgG2b; Washington
State University
Monoclonal
Center, Pull◦ C for 30 min. After washing with PBS containing 1% bovine serum albumin
USA)
at
25
man, WA, USA) at 25 °C for 30 min. After washing with PBS containing 1% bovine serum(BSA;
Sigma–Aldrich,
St. Louis,
cells werewith Alexa
FluorFluor
647albumin(BSA; Sigma–Aldrich,
St. MO,Louis,USA),MO, USA),cells stained
were stainedwith Alexa
conjugatedanti-rabbit
IgG(H+L)
goat
F(ab’)2(ThermoFisherScientific),
FITC-conjugated647-conjugated anti-rabbit IgG(H+L) goat F(ab’)2 (Thermo Fisher Scientific), FITC-conjuanti-mouse
IgG2bIgG
goat
F(ab’)2 (Beckman Coulter, Fullerton, CA, USA), PerCp/Cy5.5gated anti-mouse
2b goat F(ab’)2 (Beckman Coulter, Fullerton, CA, USA), PerCp/Cy5.5conjugatedanti-bovine
CD3(MM1A; Washington
Washington State
University Monoclonal
Monoclonal AnAnconjugated anti-bovine CD3 mAb
mAb (MM1A;
State University
tibody Center), PE/Cy7-conjugated anti-bovine CD4 mAb (CC8; Bio-Rad, Hercules, CA,tibody Center), PE/Cy7-conjugated anti-bovine CD4 mAb (CC8; Bio-Rad, Hercules, CA,USA), PE-conjugated anti-bovine CD8 mAb (CC63, Bio-Rad), and Fixable Viability DyeUSA), PE-conjugated anti-bovine CD8 mAb (CC63, Bio-Rad), and Fixable Viability Dye
eFluor 780 (Thermo Fisher Scientific) at 25 ◦ C for 30 min. MM1A and CC8 mAbs were
eFluor 780 (Thermo Fisher Scientific) at 25 °C for 30 min. MM1A and CC8 mAbs wereconjugated with PerCp/Cy5.5 and PE/Cy7, respectively, using Lightning-Link Conjugation
conjugated with PerCp/Cy5.5 and PE/Cy7, respectively, using Lightning-Link ConjugaKits (Abcam). Cells were then washed and immediately analyzed using FACSLyric (BDtion Kits (Abcam). Cells were then washed and immediately analyzed using FACSLyric
Biosciences) and FACSuite software (BD Biosciences).
(BD Biosciences) and FACSuite software (BD Biosciences).
2.3. PBMC Cultivation Assay
T-cell responses to vaccination were analyzed using a cultivation assay of PBMCs.
Fresh PBMCs (1 × 106 ) were cultured with 5 × 106.0 TCID50 /mL of UV-inactivated BRSV(NMK7 strain), 5 × 106.0 TCID50 /mL of UV-inactivated BVDV-1a (No.12 strain), or 5 µg/mL
of concanavalin A (Con A; Sigma–Aldrich) in triplicate in 96-well round-bottom microplates(Corning Inc., Corning, NY, USA) containing 100 µL of RPMI1640 Medium (Sigma–Aldrich)
supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), 100 IU/mL of
penicillin, 100 µg/mL of streptomycin, and 0.01% L-glutamine (Thermo Fisher Scientific)
at 37 ◦ C with 5% CO2 for seven days. After cultivation, cells were harvested and used forflow cytometric analysis. Culture supernatants were collected and stored at −30 ◦ C. IFN-γ
concentrations in supernatants were measured using ELISA (Bovine IFN-γ ELISA Basic Kit;
Mabtech, Nacka Strand, Sweden) in duplicate according to the manufacturer’s protocol.

Vaccines 2023, 11, 559

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Cultured PBMCs were collected and incubated in PBS containing 10% goat serum(Thermo Fisher Scientific) as described above. Cells were then washed and labeled with
Alexa Fluor 488-conjugated anti-bovine CD25 mAb (CACT116A; VMRD, Pullman, WA,USA), Alexa Fluor 647-conjugated anti-bovine CD69 mAb (KTSN7A; Washington State
University Monoclonal Antibody Center), PerCp/Cy5.5-conjugated anti-bovine CD3 mAb(MM1A; Washington State University Monoclonal Antibody Center), PE/Cy7-conjugatedanti-bovine CD4 mAb (CC8; Bio-Rad), PE-conjugated anti-bovine CD8 mAb (CC63, BioRad), and APC/Cy7-conjugated anti-TCR1-N24 mAb (GB21A; Washington State University
Monoclonal Antibody Center) for 20 min at 4 ◦ C. MM1A, CC8, and GB21A mAbs wereconjugated with corresponding fluorochromes using Lightning-Link Conjugation Kits (Abcam). CACT116A and KTSN7A mAbs were prelabeled with Zenon Mouse IgG1 Labeling
Kits (Thermo Fisher Scientific). Cells were then washed with 1% BSA-PBS and immediately
analyzed using FACSVerse (BD Biosciences) and FACSuite software (BD Biosciences).
2.4. Statistical Analyses
All statistical tests were performed in GraphPad Prism 6 (GraphPad Software, San
Diego, CA, USA). p-values less than 0.05 were considered statistically significant.
3. Results
3.1. Kinetics of PD-1 Expression by T Cells in Vaccinated Calves
PD-1 expression on T-cell subsets was analyzed by flow cytometry of PBMCs isolated
from calves before and after vaccinations with Calfwin 6 with or without the administrationof anti-PD-L1 chAb (Figure S1). There was a trend toward an increased proportion of
PD-1+ cells in each T-cell subset after primary and booster vaccinations (Figure S2A–C). In
particular, PD-1 expression was upregulated in CD3+ CD4+ , CD3+ CD8+ , and CD3+ γδTCR+
T cells at day 49 after booster vaccination in calves that received the vaccine only (V group),
compared with before the booster vaccination (at day 42; Figure 2A–C). In contrast, no
upregulation of PD-1 expression was observed in any T-cell subset in vaccinated calvestreated with anti-PD-L1 Ab (VA group) at day 49 (Figure 2A–C). Thus, two-dose vaccination with Calfwin 6 induced a higher proportion of PD-1+ exhausted T cells5inofcalves.
Vaccines 2023, 11, x FOR PEER REVIEW
13
These findings indicate that the PD-1/PD-L1 pathway may inhibit the induction of T-cell
responses following vaccination in calves.

FigureKineticsofofPD-1
PD-1expression
expression
T cells
beforevaccination.
Proportions
Figure 2.
2. Kinetics
in in
T cells
beforeandandafterafter
vaccination.
(A–C)(A–C)
Proportionsof+ cells+CD4+ (A),+CD3+CD8+ ++γδTCR+ +T cell populations
+
+
+
+
PD-1
in
CD3
(B),
and
CD3
(C)
beforeof PD-1 cells in CD3 CD4 (A), CD3 CD8 (B), and CD3 γδTCR T cell populations(at(C)daybefore42)dayand42)
after
booster(at day 49).
each subpopulation
was analyzed was(at
andafter vaccination
booster vaccination(atPD-1dayexpression
49). PD-1 inexpressionin each subpopulation
by flow cytometry using PBMCs isolated from calves administered Calfwin 6 only (circle, n = 6) oranalyzed by flow cytometry using PBMCs isolated from calves administered Calfwin 6 only (circle,
Calfwin 6 and anti-PD-L1 Ab (diamond, n = 6). Significant differences between each pair of groups
nwere= 6) determinedor Calfwin 6using
and anti-PD-L1
Ab (diamond,= 6). Significant
differences
betweenand
eachreppair ofthe Mann–Whitney
U test. nn.s.—notsignificant.
Gating strategies
groupsweredot
determinedthe cytometricMann–Whitney
U test.
n.s.—notsignificant.
resentative
plots fromusing
the flow
analysisare shownin FigureS1. Gating strategies andrepresentative dot plots from the flow cytometric analysis are shown in Figure S1.

3.2. Activation of T Cells by the Combination of Vaccination and PD-L1 Blockade in Calves
3.2. Activation of T Cells by the Combination of Vaccination and PD-L1 Blockade in Calves
To evaluate the adjuvant effect of the combinatorial use of anti-bovine PD-L1 antievaluate
the adjuvant
effect of theuse of antigens
anti-bovine
PD-L1
antibody
bodyToandvaccination,
T-cell responses
to combinatorial
BRSV and BVDV-1
were
analyzed.
andresponses
to BRSV
BVDV-1
antigens
were
analyzed.
First,vaccination,
PBMCs wereT-cellstimulatedwith BRSV
and and
BVDV-1
antigens
and theexpression
levelsFirst,
PBMCs
were
stimulated
with
BRSV
and
BVDV-1
antigens
andtheexpression
levelsof CD25 and CD69, as activation-induced markers (AIM) [17], were analyzed
in of
+
+
+
+
+
+
CD3 CD4 , CD3 CD8 , or CD3 γδTCR T cells by flow cytometry (Figure S3). After booster
vaccination (at day 70), CD3+CD4+, CD3+CD8+, and CD3+γδTCR+ T-cell responses to BRSVor BVDV-1 antigen were observed, with antigen stimulation increasing the population of
CD25+CD69+ cells in each T-cell subset of cultivated PBMCs (Figure S4).

Vaccines 2023, 11, 559

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CD25 and CD69, as activation-induced markers (AIM) [17], were analyzed in CD3+ CD4+ ,CD3+ CD8+ , or CD3+ γδTCR+ T cells by flow cytometry (Figure S3). After booster vaccination (at day 70), CD3+ CD4+ , CD3+ CD8+ , and CD3+ γδTCR+ T-cell responses to BRSVor BVDV-1 antigen were observed, with antigen stimulation increasing the population of
CD25+ CD69+ cells in each T-cell subset of cultivated PBMCs (Figure S4).
The frequency of CD25+ CD69+ cells was significantly increased in CD3+ CD4+ T cells
stimulated with BRSV and BVDV-1 in the VA group after the booster vaccination at day
70 (Figure 3A). In addition, the frequency of AIM+ cells (CD25+ CD69+ , CD25+ CD69− ,and CD25− CD69+ cells) in the CD3+ CD4+ T-cell population was increased in stimulated
PBMCs in the VA group at day 70 (Figure 3B). The AIM+ responses of CD4+ T cells to
BVDV-1 antigen were also significantly enhanced at day 28 after primary vaccination(Figure 3B). The kinetics of CD4+ T-cell activation demonstrated strong induction after thebooster vaccination in the VA group. On the other hand, CD4+ T-cell activation was notstrongly induced even after the booster vaccination, except in V5 (Figure S5A,B). 6Thus,
Vaccines 2023, 11, x FOR PEER REVIEW
of 13
CD4+ T-cell responses to the vaccine were enhanced when the vaccine was combined withthe administration of anti-PD-L1 Ab in calves.

FigureCD4++T-cell
T-cellresponses
responsestotoBRSVBRSVand
andBVDV-1
BVDV-1antigens.
antigens.(A,B)(A,B)
Proportions
Figure 3.
3. Activation of CD4
Proportions
of
+
+
+
+
+
+
−
−
+
+
+
+
+
+
−
−
+
of
CD25
CD69
(CD25
, andCD69
CD25cells)
CD69
cells)
CD25
CD69
cellscells(A) (A)
andandAIMAIM
cells cells(CD25
CD69CD69, CD25, CD25
CD69 ,CD69
and CD25(B)+ inthe+ T-cell+ CD4+ T-celloffraction
CD3
fraction
PBMCsofstimulatedwith BRSV(left)or BVDV-1
antigen (right).
(B)
in+CD4
the CD3
PBMCs stimulated
with
BRSV
(left) or BVDV-1
antigen PBMCs(right).
isolatedfromcalves
that
had
been
administeredCalfwin6
only(circle,n=6)or
Calfwinand antiPBMCs isolated from calves that had been administered Calfwin 6 only (circle, n = 6) or 6Calfwin
6PD-L1
Ab (diamond,n = 6) atnday
day 28 day(gray),and day
70day(black)were cultured
in theandanti-PD-L1
Ab (diamond,= 6)14at (white),day 14 (white),28 (gray),and70 (black)were cultured
presence of BRSV or BVDV-1 antigen for seven days. The expression of CD25 and CD69 in eachin the presence of BRSV or BVDV-1 antigen for seven days. The expression of CD25 and CD69 in
subpopulation was analyzed by flow cytometry. Gating strategies and representative dot plots foreach subpopulation was analyzed by flow cytometry. Gating strategies and representative dot plots
the flow cytometric analysis are shown in Figure S3. Significant differences between groups wereforthe flow cytometricanalysis
inpFiguredeterminedusing Tukey’s
test. are* p shown< 0.05. **< 0.01.S3. Significant differences between groups were
determined using Tukey’s test. * p < 0.05. ** p < 0.01.

We then evaluated the activation of the other T-cell subsets, including CD8++ andWe+ then evaluated the activation of the other T-cell
subsets, including CD8 andγδTCR T cells, before and after vaccination. In the CD3+CD8++ T-cell population of BRSVγδTCR+ T cells, before and after vaccination. In the CD3+ CD8
T-cell population of BRSVand BVDV-1-stimulated PBMCs, the proportion of CD25+CD69+ cells was significantly inand BVDV-1-stimulated PBMCs, the proportion of CD25+ CD69+ cells was significantly
creased in the VA group after primary vaccination at day 28 (Figure 4A). In the V group,
the frequency of CD25+CD69+CD8+ cells was increased following BVDV-1 stimulation but
not BRSV stimulation (Figure 4A). Similar patterns were observed for the proportion of
AIM+CD8+ T cells (Figure 4B). In particular, the frequency of BRSV-reactive AIM+CD8+ T
cells was significantly higher in the VA group compared with the V group at day 28 (Fig-Vaccines 2023, 11, 559

6 of 12

increased in the VA group after primary vaccination at day 28 (Figure 4A). In the V group,
the frequency of CD25+ CD69+ CD8+ cells was increased following BVDV-1 stimulation but
not BRSV stimulation (Figure 4A). Similar patterns were observed for the proportion of
AIM+ CD8+ T cells (Figure 4B). In particular, the frequency of BRSV-reactive AIM+ CD8+
T cells was significantly higher in the VA group compared with the V group at day
Vaccines 2023, 11, x FOR PEER REVIEW
7 of 28
13(Figure 4B). These results indicate that more than one administration of the vaccine combined with PD-L1 blockade can also induce CD8+ T-cell responses to the vaccine.

Figure 4.
4. Activation
Activation ofof CD8CD8++T-cell
T-cellresponses
responses following
following vaccination
vaccination andand PD-L1
PD-L1 blockade.
blockade. (A,B)(A,B) ProProFigure++ cells (CD25+ CD69+ , CD25+ CD69− , and CD25−++
+ cells+, CD25+CD69−, andportionsof CD25
CD25+CD69
CD69++cells
cells(A)(A)andandAIMAIM
portions of(CD25+CD69
CD25−CD69CD69
cells)++
cells)
the+CD8
CD3+ T-cellCD8 fraction
T-cell fractionof PBMCs
stimulatedwith(left)
BRSV
or BVDV-1(B)
in (B)
the inCD3
of PBMCs
stimulatedwith BRSVor (left)
BVDV-1
antigenantigen(right).
PBMCs isolated
from calvesthat
had that
beenhadadministeredCalfwin 6Calfwin
only (circle,n =(circle,6) or Calfwin
6(right).
PBMCs isolated
fromcalvesbeen administered
6 onlyn = 6) orandanti-PD-L1
Ab (diamond,n = 6) on dayday
28 (gray),day 70and(black)were
culCalfwin
6 and anti-PD-L1
Ab (diamond,n = 146) (white),on day 14(white),day and
28 (gray),day 70(black)turedcultured
in the presenceof BRSVoforBRSVBVDV-1
antigenantigen
for sevenexpressionof CD25
CD69
werein the presence
or BVDV-1
fordays.
sevenThedays.
The expressionof andCD25
andin each subpopulation was analyzed by flow cytometry. Gating strategies and representative dot
CD69 in each subpopulation was analyzed by flow cytometry. Gating strategies and representative
plots for flow cytometric analysis are shown in Figure S3. Significant differences among groups
dot plots for flow cytometric analysis are shown in Figure S3. Significant differences among groups
were determined using Tukey’s test. * p < 0.05. *** p < 0.001.
were determined using Tukey’s test. * p < 0.05. *** p < 0.001.

Inγδ T
T cells
are thethe most
most abundant
abundant subset
subset ofof TT cells
cells inin blood
blood [18].
[18]. γδIn calves,
calves, γδcells areγδ T
T cells
cells
have
also
been
reported
tobeinvolvedinvaccineresponses
aseffectorcells
incattle[19–
have also been reported to be involved in vaccine responses as effector cells in cattle [19–21],
21],
makingthe analysis
γδ essential
T cells essential
for immunologicalof vaccine
makingthe analysisof γδ Tofcells
for immunologicalevaluationsevaluationsof vaccine programs
inprograms
incalves.
AIM
analysisbyflowcytometrydemonstrated
that
thefrequenciesof+calves. AIM analysis by flow cytometry demonstrated that the frequencies of CD25 CD69+ cells+CD69+ cells and AIM+ cells ++γδTCR+ T-cell population were significantly
CD25
inT-cellthe CD3+ cellsand AIMin the CD3+γδTCR
population
were significantly increased in PBMCs
increasedin
PBMCs
stimulated
with
BRSV
and
BVDV-1
the
VA groupat day70atcomstimulated with BRSV and BVDV-1 in the VA group at dayin70
compared
with
those
day
+γδTCR+ T+
+
pared
with
those
at
day
14
(Figure5A,B).
In
the
V
group,
fractions
in
the
CD3
14 (Figure 5A,B). In the V group, fractions in the CD3 γδTCR T-cell population stimulated
cell
population
byincreased
BVDV-1 at
wereat day 70 with
but not
inby BVDV-1
werestimulated
significantly
daysignificantly
70 but not in increased
the cells stimulated
BRSV
the
cells
stimulated
with
BRSV
(Figure5A,
B).
Althoughtheantigenspecificityofγδ
T(Figure 5A, B). Although the antigen specificity of γδ T cells during vaccination remains unclear,cells
duringvaccination
remains
unclear,
we
found
that
γδ
T
cells
are
also
involvedinthe
we found that γδ T cells are also involved in the immune response to this vaccination, andimmuneresponsewas
to enhanced
this vaccination,
andthat this
response
wasAIMenhancedby PD-L1
that this response
by PD-L1
blockade.
Taken
together,assays usingflow
blockade. Taken together, AIM assays using flow cytometry demonstrated that the combination of vaccination and PD-L1 inhibition enhanced the activation of vaccine-responsive T cells.

Vaccines 2023, 11, 559

7 of 12

Vaccines 2023, 11, x FOR PEER REVIEW

8 of 13

cytometry demonstrated that the combination of vaccination and PD-L1 inhibition enhanced
the activation of vaccine-responsive T cells.

Figurefollowing
vaccination
andand
PD-L1
blockade.
(A,B)
ProporFigure5.5.Activation
Activationofofγδ
γδT-cellT-cellresponses
responses
following
vaccination
PD-L1
blockade.
(A,B)
Pro+ CD69+ cells+ cells+ CD69+ , CD25+ CD69− , −and CD25−−CD69++ cells)
tions
of
CD25
(A)
and
AIM
(CD25
+
+
+
+
+
+
portions of CD25 CD69 cells (A) and AIM cells (CD25 CD69 , CD25 CD69 , and CD25 CD69 cells)
+
+
(B)
(B)in
inthe
theCD3CD3+γδTCR
γδTCR+ T cells fraction of PBMCs stimulated with BRSVBRSV (left)(left) oror BVDV-1
BVDV-1 antigen
antigen(right).
(right). PBMCs isolated from calves thatthat had
had been
been administeredadministered Calfwin
Calfwin 66 only
only (circle,(circle, nn == 6)6) oror
Calfwin6
andanti-PD-L1
Ab(diamond,
n
=
6)
on
day
14
(white),day
28(gray),
andday
70(black)Calfwin 6 and anti-PD-L1 Ab (diamond, n = 6) on day 14 (white), day 28 (gray), and day 70 (black)werecultured
culturedininthe
thepresence
presence
BRSV
BVDV-1
antigenfor sevendays.
expressionof CD25
were
ofof
BRSV
or or
BVDV-1
antigenfor sevendays.
The Theexpressionof CD25
and
and
CD69
in
each
subpopulation
was
analyzedbyflow
cytometry.
Gatingstrategies
andrepresentaCD69 in each subpopulation was analyzed by flow cytometry. Gating strategies and representative
tive dot plots for flow cytometric analysis are shown in Figure S3. Significant differences among
dot plots for flow cytometric analysis are shown in Figure S3. Significant differences among groups
groups were determined using Tukey’s test. * p < 0.05. ** p < 0.01.
were determined using Tukey’s test. * p < 0.05. ** p < 0.01.

3.3. Enhancement
Enhancement ofof IFN-γ
IFN-γ Response
Response by
by thethe Combination
Combination ofof Vaccination
Vaccinationand
and PD-L1
PD-L1Blockade
Blockade
3.3.
in
in Calves
CalvesTh1Th1 cytokine
cytokine responses
responses toto thethe vaccine
vaccine were
were examinedexamined as
as aa functional
functional parameterparameter ofof TT
cells.
byby
antigenstimulationwere
measuredascells. In
Inthisthisexperiment,
experiment,IFN-γ
IFN-γresponses
responsesinduced
inducedantigenstimulationwere
measuredaasrepresentationofTh1
cytokine
responses.
PBMCs
from
calvesvaccinatedor
unvaccinateda representation of Th1 cytokine responses. PBMCs from calves vaccinated or unvacwith
Calfwinwere cultured
the presence
BRSV orofBVDV-1
antigenandantigen
anti-PD-L1
cinatedwith 6Calfwin
6 were inculturedin theofpresence
BRSV or
BVDV-1
and
Ab,
with
IFN-γ
production
measuredby
ELISA.
There
was
a
trend
towardincreased
IFN-γ
anti-PD-L1 Ab, with IFN-γ production measured by ELISA. There was a trend toward
responses
antigens
following
PD-L1 blockadein vaccinatedanimals(Figureanimals
S6A,B).
increased to
IFN-γ
responses
to antigens
following
PD-L1 blockade
in only
vaccinatedThisfinding
indicatesthat
thisvaccineincalvesincreased
theproportionoffunctionally
only (Figure S6A,B). This finding indicates that this vaccine in calves increased the proexhausted
populations
via activationof the PD-1/PD-L1
pathway.
portion ofT-cellfunctionally
exhausted
T-cell populations
via activationof the PD-1/PD-L1
Comparing
IFN-γ
responses
incalvestreatedwithanti-PD-L1
Ab before vaccination
pathway.
and after
the booster
vaccination,
wastreateda trendwith
towardincreasedtoComparing
IFN-γ
responses there
in calvesanti-PD-L1
AbIFN-γ
beforeresponses
vaccination
BRSV in the VA group at day 70 (Figure 6A). The mean concentration of IFN-γ was 0.02 andand after the booster vaccination, there was a trend toward increased IFN-γ responses to
1.05 ng/mL in BRSV-stimulated PBMCs in the V and VA groups, respectively (Figure 6A).
BRSV in the VA group at day 70 (Figure 6A). The mean concentration of IFN-γ was 0.02
Furthermore, the IFN-γ response to BVDV-1 stimulation was significantly higher in the VA
and 1.05 ng/mL in BRSV-stimulated PBMCs in the V and VA groups, respectively (Figuregroup at day 70 (mean, 8.16 ng/mL) compared with day 14 (mean, 1.71 ng/mL; Figure 6B),
6A). Furthermore, the IFN-γ response to BVDV-1 stimulation was significantly higher inindicating that a Th1 cytokine response to the antigen was induced by the combinatorial
the VA group at day 70 (mean, 8.16 ng/mL) compared with day 14 (mean, 1.71 ng/mL;use of vaccination and PD-1/PD-L1 blockade. In summary, these results demonstrate that
Figure 6B), indicating that a Th1 cytokine response to the antigen was induced by the
combinatorial use of vaccination and PD-1/PD-L1 blockade. In summary, these results

Vaccines 2023, 11, x FOR PEER REVIEW
Vaccines 2023, 11, 559

9 of 13
8 of 12demonstrate that combinatorial treatment with vaccination and anti-PD-L1 Ab functioncombinatorial treatment with vaccination and anti-PD-L1 Ab functionally activates T cells
ally activates T cells and enhances Th1 cytokine responses to antigen in vaccinated calves.
and enhances Th1 cytokine responses to antigen in vaccinated calves.

FigureActivation
IFN-γ
responses
following
vaccination
and
PD-L1
blockade.
(A,B)
IFN-γ
Figure
6. 6.
Activation
of of
IFN-γ
responses
following
vaccination
and
PD-L1
blockade.
(A,B)
IFN-γ
responses
to
BRSV
(A)
or
BVDV-1
antigen(B)
beforevaccination
(on
day
14)
or
after
boost
vaccinaresponses to BRSV (A) or BVDV-1 antigen (B) before vaccination (on day 14) or after boost vaccination70). PBMCs
isolatedfrom calves
thatbeen
hadadministeredbeen administeredCalfwin
only n(left,= 6)(attionday(at70).day
PBMCs
isolatedfrom calves
that had
Calfwin6 only6 (left,= 6)noror Calfwin 6 and anti-PD-L1 Ab (right, n = 6) on days 14 and 70 were cultured in the presence ofCalfwin 6 and anti-PD-L1 Ab (right, n = 6) on days 14 and 70 were cultured in the presence of BRSVBRSV or BVDV-1 antigen for seven days. IFN-γ production from PBMCs isolated from each animal
or BVDV-1 antigen for seven days. IFN-γ production from PBMCs isolated from each animal was
was measured by ELISA in duplicate. Significant differences between groups were determined usmeasured
by ELISA signed-rankin duplicate.sum
Significant
ing the Wilcoxon
test. differences between groups were determined using the
Wilcoxon signed-rank sum test.

Discussion
4. 4.
Discussion
Calfpneumoniahas
hasbeenbeenthethegreatest
greatestcause
causeofof economic
economic loss
loss inin thethe cattle
Calfpneumonia
cattle industry
indusIBRV,and
andPI3VPI3Vcause
causeprimaryprimaryinfection
infection
calvestry[22,23].
[22,23].Viruses
Virusessuch
suchasasBRSV,
BRSV, BVDV,
BVDV, IBRV,
inincalvesleading
totheaccumulationofenvironmental
stress[24].
Secondaryandtertiary
bacterial
leading to the accumulation of environmental stress [24]. Secondary and tertiary bacterial
infections
maythendevelop,
resultingseveremixed
infections
that
lead
death
infections
maythendevelop,
resultinginin
severemixed
infections
that
lead
toto
death
oror
lifelong
growthfailurein
infected
calves
[24].
As
a
first
line
of
defenseagainstrespiratory
lifelong growth failure in infected calves [24]. As a first line of defense against respiratory
infections
calves,multivalentvaccines
againstviralpathogenshave
beendeveloped
infections
in incalves,multivalentvaccines
againstviralpathogenshave
beendeveloped
andincorporated
into
calf
vaccination
programs[25–27].However,
However,somecalvesstill
still
deand
incorporated
into
calf
vaccination
programs[25–27].
somecalvesdevelopinfections
despiterepeated
vaccinations,indicating
insufficientinductionof adapvelop
infections
despiterepeated
vaccinations,indicating
insufficientinductionof adaptive
tive immune
responses.
In the development
and evaluationof vaccines,
the induction
immuneresponses.
In the development
and evaluationof vaccines,
the inductionof neu-of
neutralizing
antibodies
is
the
most
importantfactor,andtheanalysisofT-cellresponses
tralizing antibodies is the most important factor, and the analysis of T-cell responses is often
is often neglected[27–31]. Accordingly,
aredatalimiteddata regarding
the induction
neglected[27–31]. Accordingly,
there are there
limitedregardingthe inductionof T-cell ofT-cell responses
following
the administrationof commercially
responses
following
the administrationof commerciallyavailableavailable
vaccines.vaccines.
Immunotherapy
targetingPD-1/PD-L1pathway
pathwayhas
has
been
widelyappliedImmunotherapy
targetingthethePD-1/PD-L1
beenwidelyappliedin inthetheclinicaltreatmentof humantumors.
PD-1/PD-L1
blockadestudiedand declinicaltreatmentof humantumors.
PD-1/PD-L1
blockadehas hasalsoalso
beenbeenstudiedand develvelopedin veterinary
medicine
as a novel
treatment
for canine
and infectious
disopedin veterinary
medicine
as a novel
treatment
for canine
tumorstumors
and infectious
diseasesin domestic[11–13,32–36].
In addition,clinicaltrialsof the combinatorial
in easesdomestic
animals animals[11–13,32–36].
In addition,clinical trialsof thecombinatorial
use oftherapeutic
vaccines vaccines
against tumors
PD-1/PD-L1
inhibitorsinhibitors
are currently
underway
use of therapeutic
againstandtumorsand PD-1/PD-L1
are currently
unin derway
humansin[37].
However,
there are currently
reported
combining
vaccination
humans[37]. However,there are no
currently
nostudies
reportedstudies combining
vacandPD-1/PD-L1
blockade inblockade
veterinary
medicine. In
addition,Inthere
are limited
reported
cinationand PD-1/PD-L1
in veterinary
medicine.
addition,
there arelimitedstudiesof thecombinedof live-attenuatedvaccines against
viruses
andviruses
PD-1/PD-L1
reportedstudiesof theusecombineduse of live-attenuatedvaccines
againstand PDinhibitors.
the present
study,
we addressedtheseissues byvalidating
thevalidating
immunological
1/PD-L1 In
inhibitors.
In thepresent
study, weaddressed
theseissues by
the imeffectofthecombinationofvaccination
andanti-PD-L1
Ab
inabovinevaccinemunological effect of the combination of vaccination and anti-PD-L1 Ab in amodel.
bovine vacWe
observed an increased proportion of PD-1+ cells in all T-cell subsets after vaccine
model.
+ cells in after
cination
inobserved
calves. This
upregulation
was moreprominent
boostervaccinationinWe
an increased
proportionof PD-1
all T-cell
subsets
after vaccinathetion
V groupbut not
the VA group,
indicating
T-cell activationand cytokine
responses
in calves.
Thisinupregulation
wasmore prominent
after booster
vaccinationin the V
wereenhancedboostervaccinationin the
VA activationgroup. In addition,slightdifferences
group
but notafterin the
VA group,
indicating
T-celland cytokine
responses
were+ , CD8+ , and γδTCR+ T-cellin enhanced
responses after
to PD-L1
blockadewereobserved
betweenCD4
booster vaccination in the VA group. In addition, slight differences inVaccines 2023, 11, 559

9 of 12subsets, including increases in the rate and potency of T-cell activation. Although PD-1
expression levels and responsiveness to PD-L1 blockade may be correlated in T-cell fractions, no clear difference was observed due to the limited number of animals includedin the present study (data not shown). The specific T-cell fractions, such as memory or
effector T-cell subpopulations, responsible for mediating the effects of PD-L1 inhibition
remain unclear. To determine the mechanism underlying the induction of T-cell responses
following the vaccination with attenuated viruses, phenotypic and transcriptomic analyses
of T cells following combinatorial use of vaccination and PD-L1 blockade are warranted.
In the present study, we evaluated T-cell responses using AIM assays and cytokine
measurements. The AIM assay is a technique used to evaluate vaccination efficacy inhumans by measuring multiple markers of T-cell responses [17,38]. Furthermore, numbersof AIM+ T cells is known to be correlated with numbers of cytokine-producing T cells
in response to vaccine antigens [17]. In the present study, IFN-γ was measured as arepresentative cytokine produced by effector T cells. The results of the AIM and IFN-γ
assays were consistent. Although the AIM assay is not widely used in bovine immunology,
this technique may have utility in further studies of T-cell responses to vaccination in cattle.
T-cell responses to two viral antigens in the hexavalent vaccine, BRSV and BVDV1, were examined in the present study and found to be inhibited by the PD-1/PD-L1
pathway after the vaccination, although some differences were observed in the responses
produced by PD-L1 inhibition. The induction of T-cell responses in addition to humoral
responses is considered important for complete protective immunity against BRSV and
BVDV [25,26]. In RSV infection, IFN-γ is a key cytokine that mediates the antiviral Th1
responses that activate natural killer cells and cytotoxic CD8+ T cells, thereby resulting inviral clearance and recovery from a viral challenge in calf and mouse models [39–43]. On
the other hand, BVDV-specific CD4+ and CD8+ T cells play essential roles in protecting
against BVDV infection cattle in the absence of a BVDV-neutralizing Ab response [44–47].
However, the present study did not examine the protective effects of the combinatorial use
of vaccination and PD-L1 blockade against viral challenge. Viral challenge experiments
should be conducted to further validate the efficacy of PD-L1 blockade as an adjuvant tovaccination in calves.
In conclusion, the results of the present study demonstrate the potential utility of
PD-1/PD-L1 inhibitors as vaccine adjuvants in cattle. The combination of viral vaccination
and PD-L1 blockade may enhance vaccine efficacy and reduce the number of calf deaths
due to pneumonia and diarrhea, thereby improving dairy and beef cattle productivity. In
addition, T-cell exhaustion via the PD-1/PD-L1 pathway is a conserved immune regulatory
mechanism from domestic and companion animals to humans [2,5,6,48–54]. Accordingly,
this strategy may increase the efficacy of vaccination programs across a wide range ofanimal species, not just in cattle. Further studies using other animal species and vaccines are
required to determine the potential efficacy of PD-1/PD-L1 inhibitors as vaccine adjuvants.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vaccines11030559/s1, Figure S1: Gating strategy and representative
dot plots for expression analyses of PD-1 in the vaccinated calves; Figure S2: Kinetics of PD-1
expression in T cells before and after vaccination; Figure S3: Gating strategy and representative
dot plots for the AIM assay in the vaccinated calves; Figure S4: Response of T cell subsets against
antigen stimulations after vaccination; Figure S5: Kinetics of activation status of T cells before andafter vaccination; Figure S6: Effect of PD-L1 blockade on IFN-γ response to viral antigens.
Author Contributions: Conceptualization, T.O., S.K. and K.O.; methodology, T.O. and S.K.; validation, T.O., S.K., N.M. and S.M.; formal Analysis, T.O.; investigation, T.O., H.N. (Hayato Nakamura),
O.G., Y.S. (Yamato Sajiki), K.W., H.N. (Haruka Noda) and M.H.; resources, H.N. (Haruka Noda), M.H.,
Y.K. and Y.S. (Yasuhiko Suzuki); data Curation, T.O. and S.K.; writing—original draft preparation,
T.O. and S.K.; writing—review and editing, M.H., Y.K., Y.S. (Yasuhiko Suzuki), N.M., S.M. and K.O.;
visualization, T.O.; supervision, S.K. and K.O.; project administration, S.K.; funding acquisition, T.O.,
Y.K. and S.K. All authors have read and agreed to the published version of the manuscript.

Vaccines 2023, 11, 559

10 of 12Funding: This work was supported by JSPS KAKENHI grant number 19KK0172, 22H02503, 22K19232[to S.K.], 19K15993, and 22K15005 (to T.O.), grants from the Project of the NARO, Bio-oriented
Technology Research Advancement Institution (Research Program on Development of Innovative
Technology 26058 BC (to S.K.) and Special Scheme Project on Regional Developing Strategy, Grant
16817557 (to S.K.)), grants from Regulatory Research Projects for Food Safety, Animal Health andPlant Protection (JPJ008617.17935709 (to S.K.)) and Support System for the Collaborative Research ofYoung Researchers for the Next Generation by Hokkaido University (to T.O.), and supported by theJapan Agency for Medical Research and Development (AMED) under Grants JP223fa627005 (to S.K.),
JP21am0101078 (to Y.K.) and JP22ama121008 (to Y.K.). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
Data Availability Statement: The data presented in this study are available on reasonable requestfrom the corresponding author.
Acknowledgments: We are grateful to Hideyuki Takahashi, Yasuyuki Mori and Tomio Ibayashi
for their valuable advice and discussions, Kyoto Biken Laboratories Ltd. for providing the vaccine
product and viral antigens, and the staffs of Hokkaido Research Station and Snow Brand Seed Co.,
Ltd. for the excellent care and handling of animals.
Conflicts of Interest: Y.S., S.K., T.O., N.M., Y.S., K.O. and S.M. have a patent pending for materials
and techniques described in this paper (Japanese patent, application number 2018-99704). H.N. and
H.M. were employed by Snow Brand Seed Co., Ltd. The other authors declare no competing interests.

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