Salmonella Typhimurium, the major causative agent of foodborne illness inactivated by a phage lysis system provides effective protection against lethal challenge by induction of robust cell-mediated immune responses and activation of dendritic cells

Abstract

Salmonella Typhimurium infection via foodborne transmission remains a major public health threat even in developed countries. Vaccines have been developed to reduce the disease burden at the pre-harvest stage, but the cell-mediated immune response against intracellular invasion of the pathogen is not sufficiently elicited by conventional killed Salmonella vaccines, which are safer than live vaccines. In this study, we developed a genetically inactivated vaccine candidate by introducing lysis plasmid pJHL454 harboring the λ phage holin–endolysin system into S. Typhimurium; we designated this vaccine JOL1950. In vitro expression of endolysin was validated by immunoblotting, and complete inactivation of JOL1950 cells was observed following 36 h of the lysis. Electron microscopic examinations by scanning electron microscopy and immunogold labeling transmission EM revealed conserved surface antigenic traits of the JOL1950 cells after lysis. An in vivo immunogenicity study in mice immunized with lysed cells showed significantly increased serum IgG, IgG1, and IgG2a levels. Further, we observed markedly increased in vitro cell proliferation and upregulation of Th1, Th2, and Th17 cytokines in the repulsed splenic T-cells of immunized mice. In dendritic cells (DCs) treated with lysed JOL1950, we observed a significant increase in dendritic cell activation, co-stimulatory molecule production, and levels of immunomodulatory cytokines. In addition, Th1 and Th17 cytokines were also released by naïve CD4+ T-cells pulsed with primed DCs. Lysed JOL1950 also protected against lethal challenge in immunized mice. Together, these results indicate that our vaccine candidate has great potential to induce cell-mediated immunity against S. Typhimurium by facilitating the activation of DCs.

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Won and Lee Vet Res (2017) 48:66
DOI 10.1186/s13567-017-0474-x
RESEARCH ARTICLE
Salmonella Typhimurium, the major
causative agent offoodborne illness inactivated
bya phage lysis system provides eective
protection againstlethal challenge byinduction
ofrobust cell-mediated immune responses
andactivation ofdendritic cells
Gayeon Won and John Hwa Lee*
Abstract
Salmonella Typhimurium infection via foodborne transmission remains a major public health threat even in devel-
oped countries. Vaccines have been developed to reduce the disease burden at the pre-harvest stage, but the cell-
mediated immune response against intracellular invasion of the pathogen is not sufficiently elicited by conventional
killed Salmonella vaccines, which are safer than live vaccines. In this study, we developed a genetically inactivated
vaccine candidate by introducing lysis plasmid pJHL454 harboring the λ phage holin–endolysin system into S. Typh-
imurium; we designated this vaccine JOL1950. In vitro expression of endolysin was validated by immunoblotting, and
complete inactivation of JOL1950 cells was observed following 36 h of the lysis. Electron microscopic examinations by
scanning electron microscopy and immunogold labeling transmission EM revealed conserved surface antigenic traits
of the JOL1950 cells after lysis. An in vivo immunogenicity study in mice immunized with lysed cells showed signifi-
cantly increased serum IgG, IgG1, and IgG2a levels. Further, we observed markedly increased in vitro cell proliferation
and upregulation of Th1, Th2, and Th17 cytokines in the repulsed splenic T-cells of immunized mice. In dendritic cells
(DCs) treated with lysed JOL1950, we observed a significant increase in dendritic cell activation, co-stimulatory mol-
ecule production, and levels of immunomodulatory cytokines. In addition, Th1 and Th17 cytokines were also released
by naïve CD4+ T-cells pulsed with primed DCs. Lysed JOL1950 also protected against lethal challenge in immunized
mice. Together, these results indicate that our vaccine candidate has great potential to induce cell-mediated immu-
nity against S. Typhimurium by facilitating the activation of DCs.
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Introduction
Nontyphoidal salmonellosis (NTS) attributable to food-
borne transmission poses a substantial public health
challenge worldwide [1]. e incidence of NTS infection
in the US is estimated to be 1million annually, with 400
deaths [2]. Salmonella enterica serovar Typhimurium
(S. Typhimurium) is one of the most common serotype
causing NTS infections related to human illnesses, such
as acute gastroenteritis [2]. Food can be contaminated
with the pathogen during pre-harvest, harvest, and post-
harvest periods. Severe invasive Salmonella infection
such as bacteremia and septicemia frequently occurs in
the immunocompromised population, resulting in hos-
pitalization and death [3]. e ability of Salmonella to
resist environmental stresses make it difficult to eradi-
cate the pathogen in the food chain [4]. Over the last few
years, cost-effective intervention measures to minimize
the microbial load of raw products have been explored
Open Access
*Correspondence: johnhlee@jbnu.ac.kr
College of Veterinary Medicine, Chonbuk National University, Iksan
Campus, Gobong-ro 79, Iksan 54596, South Korea

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Won and Lee Vet Res (2017) 48:66
because the use of antibiotics has been compromised
by the emergence of multidrug-resistant Salmonella [5,
6]. A recent systemic review reported that vaccination
decreased Salmonella prevalence in market-weight fin-
isher swine [7], which suggests that control measures
at the farm level can reduce the risk of food-borne sal-
monellosis. Given the importance of a “farm-to-fork”
approach for the control of zoonotic food-borne diseases
[8], development of effective vaccine candidates against
salmonellosis responsible for human illnesses could
address public health concerns about zoonotic infection
through consumption of contaminated animal meats.
Several experimental vaccines containing both inac-
tivated and live attenuated candidates have been tested
[9]. Particularly, auxotrophic mutants of S. Typhimurium
have a potential as a live attenuated vaccine candidate
against the infection [10, 11]. However, the immunomod-
ulatory effects elicited by the live attenuated vaccines
have not been sufficiently assessed; their immunogenic-
ity was found to vary depending on injection route [12,
13]. New and efficient vaccine candidates that are easy
to administer and that confer high immunogenicity are
required to help protect against food-borne salmonellosis
at the pre-harvest level.
Bacterial ghosts (BG) refer to the empty, non-living
envelopes of Gram-negative bacteria produced by the
action of the coliphage lysis gene [14]. Lysis gene expres-
sion facilitates formation of transmembrane tunnels on
the surface of the bacteria through which cytoplasm and
nucleic acids are expelled due to osmotic pressure. How-
ever, it has been reported that bacteria cell lysis mediated
by gene E is not complete [15, 16], which has raised con-
cerns about the safety of BG vaccines.
In the current study, to address this technical problem
concerning the production of BG, we adapted a holin–
endolysin component from bacteriophage λ to construct
a genetically inactivated S. Typhimurium vaccine can-
didate. Holin and endolysin act in a cooperative man-
ner to cleave peptidoglycan (PG) substrates of bacterial
cell walls [17]. Endolysin, a cell wall-degrading enzyme,
accumulates in the cytoplasm [18]. At a genetically pre-
determined time when the fatal membrane lesions were
formed by holin, endolysin proteins escape through IM
lesions and consecutively degrade the PG layers. We pre-
pared the lysis plasmid pJHL464 harboring S and R genes
encoding holin and endolysin, respectively, under con-
vergent promoter components to prepare the novel inac-
tivated S. Typhimurium vaccine strain JOL1950.
We analyzed whether the antigenic properties of the
bacterial surface components were altered following
endolysin-mediated lysis by immunogold labelling and
found that the BG had an intact bacterial surface struc-
ture. Given that a conventional killed vaccine against
salmonellosis failed to induce proper cell-mediated
immunity (CMI) [19], which is required to defend against
intracellular invasion and multiplication of the patho-
gen, we investigated the ability of our vaccine construct
to be efficiently internalized by dendritic cells (DCs),
which mediate T cell-related adaptive immunity [20].
Immunoregulatory effects and protection efficacy of
the vaccine were further evaluated invivo and invitro.
is work makes a vital contribution toward protecting
against food-borne salmonellosis.
Materials andmethods
Bacterial strains andplasmids
Bacterial strains and plasmid vectors utilized in this study
are listed in Table1. A balanced-lethal system based on
the aspartate β-semialdehyde dehydrogenase (asd) gene
was used to maintain the stability of the plasmid in an
attenuated Salmonella strain [21]. e asd gene-deleted
mutants were incubated with 50g/mL diaminopimelic
Table 1 Bacterial strains and plasmids utilized in this study
Strain/plasmids Description References
E. coli
JOL232 (χ6212) F-λ-φ80 Δ(lacZYA-argF) endA1 recA1 hadR17 deoR thi-1 glnV44 gyrA96 relA1 ΔasdA4 Lab stock
Salmonella Typhimurium
JOL 401 Wild type isolate from porcine, challenge strain Lab stock
JOL1311 Δasd, used as base vaccine strain Lab stock
JOL1950 JOL1311 containing pJHL464 This study
Plasmids
pJHL172 asd+ vector, pBR ori plasmid harboring cI857/λPR promoter, araC ParaBAD, phiX174 lysis gene E[22]
pJHL319 T-easy vector harboring E gene ghost cassette with the convergent promoter system This study
pJHL360 T-easy vector harboring the R ghost cassette with the convergent promoter system This study
pJHL464 asd+ vector, pBR ori plasmid harboring cI857/λPR promoter, araC ParaBAD, the R ghost cassette composed of S,
R and R1/Rz genes This study

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Won and Lee Vet Res (2017) 48:66
acid (DAP) in culture media at 37 °C. e asd gene-
deleted vaccine strain complemented by an asd+ lysis
plasmid (pJHL464) was grown in medium supplemented
with 0.2% -arabinose. All bacterial strains used in the
study were stored at −80°C in Luria–Bertani (LB) broth
with 20% glycerol.
Construction ofthe vaccine strain
e plasmid pJHL464 contains a 1.4kb fragment of the
R lysis cassette comprising open reading frames (ORF)
of the S and R genes encoding holin and endolysin pro-
teins, respectively, and overlapping ORF of the Rz/Rz1
genes. Convergent promoter components, namely an
arabinose-inducible promoter (ParaBAD) and thermo-
sensitive λpR promoter with a cI857 repressor system,
were used for stringent regulation of the expression of
the lysis cassette in the plasmid. For the construction,
the 1.4kb lysis cassette was digested and subcloned into
the NcoI/BamHI-digested pJHL319 plasmid which is a
T-easy vector carrying the convergent promoter compo-
nents. e lysis cassette was placed between an upstream
λpR promoter and downstream anti-sense araBAD pro-
moter controlling the sequential expression of holin and
endolysin harbored in the resultant plasmid, pJHL360.
e total 4.2-kb DNA fragment which harbors the R lysis
cassette and the dual promoter components was inserted
into BglII/XhoI digested asd+plasmid pJHL172 [22]. e
resultant plasmid pJHL464 was initially introduced into
asd E.coli JOL232 (χ6212) to overcome the instability
of the plasmid. Subsequently, the resultant plasmid was
transformed into asd S. Typhimurium strain JOL1311
by electroporation. e constructed strain was desig-
nated JOL1950. Expression of endolysin was confirmed
by Western blotting with rabbit hyperimmune sera
against endolysin following the procedure described in
a previous study [23]. Further, morphological changes of
JOL1950 following the expression of lysis genes encoded
by the pJHL464 plasmid were examined by scanning
electron microscopy (SEM) [22].
Lysis eciency
e expression of endolysin is repressed below tempera-
tures of 30°C as a result of the cI857 protein binding to
the operator region of the λpR promoter. Inactivation
of thermo-labile repressor cI857 at temperatures above
40°C results in strong expression of the lysis gene cas-
sette. In the presence of arabinose, the anti-sense RNA
produced by the ParaBAD promoter could bind to its
complementary sense RNA of the lysis genes caused by
the leaky λpR promoter, thus preventing the transla-
tion and expression of the lysis genes [24]. To exam-
ine whether the holin–endolysin system effectively
inactivated JOL1950 cells, a single colony of JOL1950 was
grown in LB broth supplemented with 0.2% -arabinose
to mid-log phase at 27 °C. e number of ghost cells
were determined by measuring optical density (OD at
600nm wavelength) when the cells were grown to mid-
log phase under the condition repressing expression of
endolysin. To generate JOL1950 ghost cells, the inocula-
tion was resuspended with LB broth to remove the arab-
inose and then incubated at 42°C in a shaking incubator
(150rpm). Lytic capacity was examined by determining
the viability of cells recovered in the culture sampled
under conditions activating expression of the R lysis
cassette. Cell viability was confirmed by the number of
colony forming units (CFU) in 100L of the cells follow-
ing the lysis procedure spreading on LB plate containing
0.2% -arabinose in triplicate. e number of living cells
were counted on the plate using a 10-fold serial dilution
method. After 36h of lysis, a pellet of completely inac-
tivated JOL1950 ghost cells was stored at −70 °C until
further processing.
Immunogold labeling
Transmission electron microscopy (TEM) was conducted
to clarify whether the expression of the R lysis cassette
in JOL1950 affected its native antigenicity following the
protocol described in a previous study [25] with slight
modification. JOL1950 cells inactivated for 24 h were
fixed with 2% paraformaldehyde and 2% glutaraldehyde
in 0.1M sodium cacodylate buffer (pH 7.4) overnight at
4°C. Fixed cells were washed with phosphate-buffered
saline (PBS) three times and then absorbed onto carbon
formvar-coated copper grids (200-meshes) for 10 min.
e cells placed onto the grid were blocked in PBS con-
taining 2% BSA for 30 min and then incubated with poly-
clonal antibody produced against JOL1311 in chicken
(1:300) as a primary antibody. Sera containing the
chicken polyclonal antibody were obtained from a white
leghorn chicken at 4weeks of age intramuscularly immu-
nized with 1×107 CFU of JOL1311 cells twice within a
2-week interval. Serum samples were obtained 14days
after the final immunization and stored at −20°C until
use. Subsequently, cells were placed on 10 nm gold-
labeled goat anti-chicken IgY (1:500; Abcam Inc., USA)
as a secondary antibody for 30min. After rinsing with
PBS three times, the grids were stained with 2% uranyl
acetate and analyzed. JOL1311 cells chemically inacti-
vated by 0.2% formalin were used as the control.
Animal experiments
All animal experimentation work was approved by the
Chonbuk National University Animal Ethics Commit-
tee (CBNU2015-00085) and was carried out accord-
ing to the guidelines of the Korean Council on Animal
Care and Korean Animal Protection Law, 2007; Article

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Won and Lee Vet Res (2017) 48:66
13 (Experiments with Animals). Five-week-old female
BALB/c mice (n=16) were randomly assigned to two
groups. Group A mice were inoculated with 108 CFU
of lysed JOL1950 cells in 100L of sterile PBS via the
subcutaneous route at weeks 0 and 2, while mice in
group B received only 100L of sterile PBS on the same
schedule. Whole blood samples were collected from
the retro-orbital sinus of mice at weeks 0, 2, 4, 6, and 8
post-inoculation (pi). Serum samples isolated from whole
blood were stored at −80 °C until assayed by ELISA.
Additional mice (n= 10) were subcutaneously inocu-
lated with JOL1950 ghost cells (1×108) and sacrificed
for splenic-T cell analysis at week 2 pi. At 8weeks fol-
lowing immunization, all mice were challenged with a
lethal dose (1×106) of a virulent S. Typhimurium strain,
JOL401, via intraperitoneal administration. Mortality was
observed in the injected mice for 11days post-challenge.
Assessment ofantibody titers byenzyme‑linked
immunosorbant assay (ELISA)
Antibody responses specific to the Salmonella outer
membrane protein (OMP) fraction were evaluated by an
ELISA following the protocol previously reported [26].
e Salmonella OMP fraction prepared from the JOL401
strain [27] (500ng/well) was coated onto ELISA microti-
ter plates (Greiner, Frickenhausen, Germany). Sera were
diluted 1:100 for examination of IgG, IgG1, and IgG2a
titers. e antibodies were detected with horseradish
peroxidase (HRP)-labeled goat anti-mouse IgG, IgG1 and
IgG2a, respectively (Southern Biotechnology, Birming-
ham, AL). Predicted serum IgG titers were determined
directly from a standard curve based on serial dilutions of
purified mouse immunoglobulins (Southern Biotechnol-
ogy, Birmingham, AL, USA) [28]. Titers of serum IgG1
and IgG2a are presented as absorbance values at 470nm.
Antigen‑specic splenic T cell‑related immune response
Splenocyte preparation
Splenocytes were aseptically isolated from immunized
and non-immunized mice at week 2 pi. e spleens sam-
pled from the mice were mashed in PBS and filtered
through a 70m cell strainer. e cells were suspended
in RPMI 1640 medium (GIBCO, Cat. No. 11875093) con-
taining 5% FCS (GIBCO, Cat. No. 10099141) and then
incubated in a humidified 37°C, 5% CO2 incubator over-
night for further experiments.
In vitro cell proliferation assay
e proliferation response following in vitro antigen
stimulation was determined by MTT colorimetric assay
with 3-(4,5-dimethyl-thiazole-2-yl)-2,5-phenyltetrazo-
lium bromide [29]. Murine splenocytes seeded in 96-well
cell culture plates (1 × 106 cells/well) were incubated
with 20L of MTT (5mg/mL) at 37°C for 4h, and then
the culture supernatant was removed, followed by addi-
tion of 100L dimethyl sulfoxide (DMSO) to each well.
Cell proliferation was determined by the absorbance at
570nm of the water-soluble MTT formazan product.
Cytokine assay
To assess immunomodulatory cytokine profiles elicited
by the vaccine candidate, mRNA expression of IL-2, IL-4,
IL-8, IL-17, IL-23, and IFN-γ was measured in splenic
lymphocytes pulsed with Salmonella-specific outer
membrane protein (OMP) antigens. Splenocytes (1×106
cells/well) were stimulated with 300ng/mL of antigens in
a 96-well plate and incubated at 37°C in a 5% CO2 incu-
bator for 48h. Total RNA was isolated from the pulsed
cell cultures using GeneAll® Hybrid-RTM (GeneAll Bio-
technology, Seoul, Korea) following the manufacturer’s
instructions. Subsequently, RNA samples were con-
verted into cDNA with the ReverTra Ace® qPCR RT kit
(FSQ-101, TOYOBO, Japan). e mRNA expression of
cytokines was measured by performing real-time reverse
transcription-polymerase chain reaction (RT-PCR) with
SYBR® Green Real-time PCR Master Mix (QPK-201,
TOYOBO, Japan). Primer pairs described in a previous
study were used [30]. Transcript levels of each cytokine
were determined using the relative fold change method
(2−(CT)) based on the expression of β-actin used as an
internal standard, and results were compared between
immunized and non-immunized groups [31].
Dendritic cell analysis
e ability of lysed JOL1950 cells to trigger a T-cell-
related immune response was assessed by dendritic cell
analysis. Differentiation of immature murine bone mar-
row-derived dendritic cells (BMDC) following in vitro
infection with JOL1950 ghosts was assessed by meas-
uring the expression of surface markers and release of
immunomodulatory cytokines. In addition, to evaluat-
ing the capacity of mature DCs pulsed with JOL1950 to
stimulate autologous naïve T-cells invitro, expression of
immunomodulatory T-cell cytokines was evaluated in
CD4+ T-cells co-cultured with primed DCs via qRT-PCR.
BMDC were aseptically isolated from female C57BL/6
mice at 6weeks of age (n=3) and cultured as described
previously with slight modifications [32]. Briefly, 1×106
cells were suspended in RPMI medium containing 10%
FBS, penicillin (10units/mL), streptomycin (10g/mL),
-glutamine (0.29 mg/mL), and cytokines (10 ng/mL
murine GM–CSF and 5ng/mL IL-4). On day 5 of cul-
ture, BMDC adhering to the bottom of the plate were
seeded in six-well cell culture plates and then stimulated
with either BG at a multiplicity of infection of 10 or LPS
(500ng/mL, serotype O127:B8, Sigma-Aldrich, St. Louis,

Page 5 of 12
Won and Lee Vet Res (2017) 48:66
MO, USA) as a positive control for 48h. On day 7, the
expression of co-stimulatory molecules such as CD40,
CD80, and MHC class II on the differentiated BMDC was
analyzed by fluorescence-activated cell sorting (FACS)
with labeling of cells with FITC anti-mouse CD11c, APC
anti-mouse CD40, APC anti-mouse CD80, and APC anti-
mouse MHC class II (all Miltenyi Biotec) as described
previously [33]. e population of DC expressing co-
stimulatory surface markers was determined in CD11c-
gated DC. Gene upregulation of cytokines related to DC
differentiation such as IL-6, IL-10, IL-12, and TNF-α was
assessed in stimulated BMDC by qRT-PCR [23]. e
capacity of BMDC primed with ghosts to differentiate
naïve CD4+ T cells into effector CD4+ T cells was fur-
ther evaluated. Immature autologous CD4+ T cells were
isolated from the splenocytes of female C57BL/6 mice
at 6weeks of age (n= 4) using a magnetic bead-based
CD4+ T cell isolation kit (Miltenyi Biotec) according to
the manufacturer’s instructions. Immature CD4+ T cells
(1×108) suspended in RPMI medium containing 10%
FBS, penicillin (10 units/mL) and streptomycin (10g/
mL) were co-cultured with 1×106 of pulsed BMDC at
37°C in a humidified 5% CO2 incubator for 24h. Release
of cytokines associated with differentiation and effector
functions of 1 and 17 (IL-12, IL-17, IL-23, and IFN-
γ) was measured by assessing transcript levels of these
cytokines by qRT-PCR [23]. All primer sets used in the
cytokine assay are described in previous studies [30, 34].
Statistical analysis
Data were analyzed using GraphPad Prism 6 software
(Graph Pad Software, Inc., San Diego, CA, USA). Non-
parametric Mann–Whitney rank sum test was applied
to determine the significance of differences between the
immunized and non-immunized groups. A P value less
than 0.05 was considered to be statistically significant.
Data are presented as mean±standard error (SE).
Results
Characterization ofthe vaccine construct JOL1950
In vitro expression of endolysin in JOL1950 was validated
by Western blotting; a distinct immunoreactive band of
~17kDa was found in lane 2 (Figure1A). No band was
detected in lane 1 (Figure1A), which contained proteins
extracted from cells grown under conditions repress-
ing the activation of the lysis cassette. Morphological
changes of JOL1950 cells after lysis were examined by
SEM. After 36 h of lysis, the cell body was elongated,
and the cell surface was partially shrunk due to inferred
osmotic pressure across transmembrane tunnels (arrow-
heads, Figure1C). Cell lysis efficiency of JOL1950 inacti-
vated by the R lysis cassette was quantitatively compared
with that of JOL1311 cells not harboring lysis genes. Cells
were grown to mid-log phase in LB broth to which ara-
binose was added at 27 °C and were then washed with
LB broth three times to remove the arabinose. Subse-
quently, washed cells were incubated in LB broth at
42°C to induce the expression of the lysis genes. After
36h of lysis, no viable cells were observed in the plate in
which JOL1950 cells were inoculated (Figure2). ese
results indicate that expression of the R lysis cassette in
the JOL1950 was regulated by a transcriptional switch
Figure1 Characterization of JOL1950 ghosts constructed by
endolysin expression. A Western blot analysis. Endolysin expressed
in JOL1950 was confirmed by rabbit anti-endolysin polyclonal
antibodies. Black arrows indicate the expected size of endolysin
(~17 kDa). Lane M, size marker; lane 1, JOL1950 grown in the pres-
ence of l-arabinose at 27 °C; lane 2, JOL1950 ghosts produced under
lysis conditions. Characterization of JOL1950 S. Typhimurium ghost
cells by scanning electron microscopy (SEM). B Intact JOL1950 cells
before lysis. C JOL1950 ghost cells after 36 h of lysis. Arrowheads
indicate lysis transmembrane tunnels.
Figure2 Lysis pattern of S. Typhimurium harboring pJHL464
(JOL1950). Cells grown to mid-logarithmic phase were inactivated
by endolysin-mediated lysis. JOL1311 not carrying pJHL464 was used
as the control. CFU counts were transformed to log base 10 values.
Data are presented as mean ± standard error (SE) of three samples.

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Won and Lee Vet Res (2017) 48:66
involving the convergent promoters, resulting in 100%
lysis during the inactivation process. TEM analysis was
used to assess possible alterations of antigenic properties
on the bacterial surface. e results suggest that more
antibody-bound colloidal gold particles may be bound to
antigens on the surface of lysed JOL1950 compared to a
chemically inactivated Salmonella strain (Figure3). is
finding implies that the structure of antigenic epitopes
of the cells were less affected by the genetic inactivation
strategy than chemical treatment.
Assessment oftotal IgG isotypes andsubclasses
Antibody responses were assessed in mice immunized
with JOL1950 ghosts. Levels of serum IgG specific
to Salmonella OMP were markedly increased in the
immunized group during the observational period after
immunization compared to the control group (P< 0.05,
Figure4). e total concentration of the serum IgG sub-
classes IgG1 and IgG2a, which are specific markers of
2 and 1 cells, respectively [35], was also significantly
augmented in immunized mice at weeks 4, 6, and 8 pi
(P<0.05, Figure4), which demonstrated that JOL1950
immunization efficiently evoked activation of 1- and
2-related immune responses.
Splenic T‑cell proliferation assay
Antigen recognition induces proliferation and differen-
tiation of naïve T-cells into effector or memory T-cells,
resulting in ensuing immune responses such as memory
responses [36]. e magnitude of T-cell proliferation was
assayed by the MTT colorimetric method in mice spleno-
cytes re-pulsed invitro with the OMP fraction. e pro-
liferation response as assessed by absorbance at 570nm
was markedly increased (P<0.001) in restimulated sple-
nocytes isolated from immunized mice (0.668± 0.059)
compared to those isolated from non-immunized mice
(0.234±0.018) (Figure5A). is proliferation of splenic
T cells in response to the antigen produced by lysed
JOL1950 cells indicates the potential of this vaccine to
induce cell-mediated immunity.
Immunostimulatory cytokine analysis
Secretion profiles of immunomodulatory cytokines
IL-2, IL-4, IL-8, IL-17, IL-23, and IFN-γ were analyzed
by RT-PCR using RNA extracted from pulsed spleno-
cytes isolated from immunized mice. Expression of
cytokine genes associated with activation of CD4+ effec-
tor T helper () cells in cell supernatants was com-
pared to baseline expression (Figure5B). At week 2 pi,
there were 7.05±1.54- and 23.73±5.82-fold increases
in mRNA levels of the 1 cell associated cytokines IL-2
and IFN-γ, respectively. Immunization also upregu-
lated mRNA expression of the 2 cell-related cytokine
IL-4 (29.67 ± 4.17-fold increase). mRNA expression
of IL-17 and IL-23, which are cytokines expressed by
17 cells [37], was increased by 19.65±8.57-fold and
27.76±11.08-fold, respectively.
In vitro analysis ofdendritic cells dierentiated bythe
construct andindirect activation ofnaïve CD4+ T cells
bythe primed dendritic cells
e capacity of JOL1950 ghosts to interact with BMDC
to induce subsequent T-cell-related immunity was eval-
uated invitro. After 48h of stimulation with the ghost
cells, distinctive morphological features such as large
veils or multiple branches was shown on the surface of
the DCs, typical signs of fully differentiated DCs (arrows,
Figure 6A). e co-stimulatory surface markers MHC-
II, CD40, and CD80 were markedly elevated in mature
BMDC pulsed with ghost cells (Figures6B and C). Lysed
Figure3 Immunogold labelling of S. Typhimurium JOL1311 inactivated by chemical treatment or JOL1950 inactivated by endolysin
expression. Bacterial cells were immunogold-labeled and negatively stained for transmission electron microscopy. Arrows indicated the 10-nm
gold particles binding the surface of cells. A Naïve JOL1311 cell, B JOL1311strain treated with 0.2% formalin, C Salmonella strain carrying pJHL464
inactivated by expression of lysis genes (JOL1950 ghosts).

Page 7 of 12
Won and Lee Vet Res (2017) 48:66
JOL1950 taken up by invitro differentiated DCs elicited
a significant increase in CD40, CD80, and MHC II pro-
duction due to the presentation of antigenic peptides to
T cells in primed DCs while the LPS stimulation only
induced a marked increase in surface expression of CD40
on the DCs compared to non-primed DCs (P < 0.05).
Differential induction of cytokines by DCs treated with
lysed JOL1950 was observed. Cytokine mRNA levels
were significantly upregulated in response to uptake of
BG (Figure6D). e mRNA expression of IL-12, which
is vital for T-cell development [38], was increased in DCs
co-cultured with ghosts or LPS. Transcript level of IL-6,
a cytokine that stimulates naïve CD4+0 cells to differ-
entiate into 17 cells [39], was markedly upregulated in
primed DCs (38.8 ± 11.3-fold increase) in comparison
to DCs treated with LPS. We assessed the invitro prim-
ing ability of DCs to evaluate whether DCs appropriately
presented the processed antigen peptides to naïve CD4+
T cell invitro and then efficiently activated CD4+ 0
into effector T cells. e differentiated CD4+ T cells were
observed after 24h of stimulation with primed DCs co-
incubated with JOL1950 ghosts (Figure 7A). Transcript
levels of immunostimulatory 1 cytokines (IL-12, IFN-
γ) and 17 cytokines (IL-17, IL-23) were significantly
elevated in naive CD4+ T cells treated with pulsed DCs
(Figure 7B). e significant increase in expression of
cytokine genes indicated that lysed JOL1950 cells effi-
ciently elicited the invitro differentiation of CD4+ T cells
into 1 or 17 phenotypes via activation of DCs.
Lethal challenge
Protective efficacy of the Salmonella ghosts was evalu-
ated by lethal challenge against S. Typhimurium infec-
tion. Mice were injected with a lethal dose of virulent S.
Typhimurium JOL401 at week 8 pi. All control mice had
died by day 3 post-infection; in contrast, all mice inocu-
lated with JOL1954 were protected against the challenge
by day 5 pi, and 3 of 8 mice survived to the end of the
observation period (Figure8).
Discussion
Salmonella Typhimurium infection, which is mainly
acquired through the uptake of contaminated food, can
cause severe medical problems, particularly in immu-
nocompromised individuals [40]. Vaccination is the
most feasible tool to counteract infection and could be
an effective pre-harvest intervention strategy to reduce
fecal shedding of the pathogen [41]. In this study, we
constructed a non-living S. Typhimurium vaccine candi-
date, JOL1950, which harbors the lysis plasmid pJHL464.
Expression of endolysin under control of convergent pro-
moters allowed stringent regulation of the transcriptional
level of the lysis genes, as validated by both invitro and
invivo studies (Figures1 and 2). is strategy prevents
leaky expression of lysis genes when cultivated under
conditions repressing these genes, which is important for
Figure4 Assessment of serum IgG antibody responses
post-immunization. Titers of serum IgG, IgG1, and IgG2a against a
Salmonella-specific antigen OMP in immunized mice were meas-
ured by ELISA assay. Mice were subcutaneously injected with lysed
JOL1950 while non-stimulated mice were inoculated with 100 μL of
sterile PBS. Data are shown as mean ± standard error (SE). NS; non-
stimulated, *P < 0.05, (vs. NS).

Page 8 of 12
Won and Lee Vet Res (2017) 48:66
optimization of the mass production of a vaccine strain
for large-scale clinical vaccine trials [22]. SEM analysis
revealed that the JOL1950 ghosts retained their original
cell morphology, but exhibited distinct surface wrinkles
due to the loss of cytoplasmic contents via lysis tunnels
around the cell body following the lysis procedure (Fig-
ure 1B). Immunogold labelling showed that JOL1950
ghosts constructed using the phage-lysis system con-
served antigenic epitopes, resulting in higher surface
antigenicity than a chemically inactivated Salmonella
strain (Figure 3). Further, robust production of serum
IgG specific to Salmonella OMP in immunized mice also
demonstrated the ability of this vaccine to elicit antibody
production against extracellular antigenic components
of JOL1950 ghosts, which were not substantially altered
during the endolysin-mediated lysis process (Figure 4).
ese results imply that the use of the holin–endolysin
system resulted in successful production of an inactivated
Salmonella vaccine, and that lysis did not negatively
affect the surface antigenic characteristics of the result-
ant JOL1950 ghosts.
Vaccine selection against Salmonella is largely an
empirical process due to a lack of understanding of how
vaccine-induced immunity affects secondary intracellular
infections. us, a promising vaccine candidate needs to
induce cell-mediated immunity, as this is critical for the
clearance of disseminated Salmonella infection. Conven-
tionally killed Salmonella vaccines are known to induce
good humoral immune responses, but poor 1 type-cell
responses [19]. Co-administration of a 1-polarizing
adjuvant chemical with a Salmonella killed vaccine was
shown to help promote a 1-related immune response,
as assessed by lymphocyte proliferation [42]. In the cur-
rent study, the inactivated vaccine candidate JOL1950
markedly increased proliferation of naïve splenic T-lym-
phocytes in response to antigen stimulation (Figure5A).
Enhanced serum levels of IgG2a and IgG1 subtypes
as markers for 1 and 2 cells, respectively, were
detected in immunized mice, indicating that immuniza-
tion with lysed JOL1950 successfully drove the differen-
tiation of naïve T cells into effector CD4+ T cell 1 and
2 subgroups. Concurrently, cytokines activating CD4+
1 T-cells and their effector cytokines, IL-2 and IFN-γ,
were secreted by stimulated splenic lymphocytes. ese
findings indicated that the inactivated JOL1950 strain
sufficiently induced cell-mediated immunity, which is
known to accelerate the clearance of secondary Salmo-
nella infection [19, 43].
Assessment of the ability of lysed JOL1950 cells to
prime immature DCs, which are potent antigen-present-
ing cells, was used to evaluate the efficacy of the vaccine
against Salmonella infection, since mature DCs differen-
tiate by engulfing foreign proteins. It has been reported
that BG produced by the expression of the PhiX174 lysis
gene results in strong activation of DCs [44, 45]. Major
antigenic components preserved on BG, which are
referred to as pathogen-associated molecular patterns
(PAMPs), are recognized by toll-like receptors (TLRs)
and other pathogen recognition receptors (PRRs), result-
ing in activation of mature DCs and subsequent induc-
tion of T-cell-related immune responses. In this study,
JOL1950 ghosts generated by the holin–endolysin lysis
system efficiently triggered adaptive immune responses
via DC-mediated antigen presentation. Expression of co-
stimulatory molecules such as CD40, CD80, and MHC
class II was significantly increased in murine BMDC
stimulated with JOL1950 ghosts in comparison to naïve
DCs (Figures 6B and C). e cytokine profile of DCs
Figure5 T-cell immune responses elicited by JOL1950 immunization. A Splenocyte proliferative responses against the OMP fraction in
immunized (group A, n = 5) mice and non-immunized (group B = 5) mice which were used as a negative control (NC) at week 2 pi determined by
absorbance values at 570 nm using the MTT assay. B Relative quantification (2−∆∆Ct) of cytokine mRNA levels in in vitro-stimulated splenic T cells
isolated from mice in the immunized group. Data are presented as scatter plots and mean ± SE of five individual values in duplicate. **P < 0.001 (vs.
restimulated NC).

Page 9 of 12
Won and Lee Vet Res (2017) 48:66
infected with ghost cells invitro also demonstrated the
immunostimulatory capacity of the JOL1950 ghosts (Fig-
ure6C). Particularly, IL-12, which enhances T cell stimu-
latory capacity [46], was expressed by BMDCs pulsed
with ghosts. e 1-derived cytokines IL-12 and IFN-γ
were produced by naïve CD4 cells cultured with primed
BMDC. ese results showed that PAMP derived from
JOL1950 ghosts were properly detected by DCs, result-
ing in induction of T-cell-related CMI to combat intra-
cellular Salmonella infection. Kirby et al. also reported
that an immune response involving DCs and cytokine
production is essential for controlling pathogen replica-
tion during the early stages of Salmonella infection [47].
Transcript levels of -17 effector cytokines IL-17 and
IL23, which are involved in adaptive immunity, were also
upregulated in CD4+ cells treated with pulsed DCs (Fig-
ure 7). Recent data demonstrated that intestinal 17
cells play a pivotal role in engulfing pathogens that cross
the epithelial mucosal barrier during the early phase of
Salmonella infection [48]. Furthermore, immunization
with JOL1950 effectively protected against a lethal dose
of virulent S. Typhimurium (Figure8). ese findings
demonstrate that JOL1950 inactivated by holin–endoly-
sin system-mediated lysis induced differentiation of naïve
T-cell into effector T-cells, which resulted in pathogen
clearance through the production of cytokines by acti-
vated DCs.
Taken together, the present results that humoral and
cellular immunity elicited by JOL1950 ghosts and their
ability to protect against infection provide compelling
Figure6 In vitro analysis of dendritic cells dierentiated by JOL1950 ghosts. Co-stimulatory molecule expression on the dendritic cell sur-
face was assessed by gating on CD11c+ cell populations by FACS. A Microscopic images of DCs captured with an inverted microscope (magnifica-
tion × 100). Representative micrographs of DC culture medium containing murine GM–CSF and IL-4 during 5 days, and DC cultures stimulated with
JOL1950 ghosts for an additional 48 h (on the 7th day of culture). DCs differentiated by stimulation displayed morphological characteristics typical
of DCs, such as the presence of dendritic processes, as indicated by the arrows. DCs supplemented with GM-CSF and IL-4 or medium only were
round in appearance or displayed veils. B Representative FACS histogram of the surface marker-positive cell population after 48 h of stimulation
with the ghost cells or LPS. C Proportion (%) of co-stimulatory molecule-positive DCs after 48 h of stimulation with the ghost cells or LPS. *P < 0.05
(vs. non-stimulated DCs). D Cytokine mRNA upregulation in DCs co-cultured with BG. *P < 0.05 (vs. cytokine mRNA upregulated in LPS-stimulated
DCs).

Page 10 of 12
Won and Lee Vet Res (2017) 48:66
evidence that JOL1950 ghosts are a promising avenue
for generating inactivated vaccines against zoonotic sal-
monellosis. e immunization induced not only T-cell-
mediated responses, but also host innate responses
mediated by DCs that were activated by antigen recog-
nition. us, given that conventional killed vaccines
of intracellular Salmonella exhibit low and inconsist-
ent protection efficacy, our genetically inactivated
holin–endolysin phage lysis system represents a rational
approach for development of an effective vaccine against
foodborne salmonellosis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GW conducted the experiments; JHL designed the experiments and JHL and
GW wrote the paper. Both authors read and approved the final manuscript.
Acknowledgements
Authors thank Ms. Song, Min Kyung, Center for University Research Facility
(CURF) at Chonbuk National University, for quality Cs-corrected-TEM image.
Funding
This work was supported by the National Research Foundation of Korea (NRF)
grant funded by the Korea government (MISP) (No. 2015R1A2A1A14001011).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 22 June 2017 Accepted: 20 September 2017
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