Vaccine 38 (2020) 3720–3728

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Oral vaccination with live attenuated Yersinia pseudotuberculosis strains

delivering a FliC180-LcrV fusion antigen confers protection against
pulmonary Y. Pestis infection
Amit K. Singh, Xiuran Wang, Wei Sun ⇑
Department of Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208, USA

a r t i c l e i n f o a b s t r a c t

Article history: We incorporated the DPfur::TT araC PBAD fur deletion-insertion mutation on top of a previous Yersinia
Received 19 February 2020 pseudotuberculosis mutant (Dasd DyopJ DyopK) to construct a new mutant designated as Yptb5, which
Received in revised form 17 March 2020 manifests the arabinose-dependent regulated delayed fur (encoding ferric uptake regulator) shut-off.
Accepted 30 March 2020
The Yptb5 strain was used to deliver an adjuvanted fusion protein, FliC180-LcrV. Levels of FliC180-
Available online 8 April 2020
LcrV synthesis were same in Yptb5 either harboring pSMV4, a p15A ori plasmid or pSMV8, a pSC101
ori plasmid containing the fliC180-lcrV fusion gene driven by Ptrc promoter. Tissue burdens of both
Keywords:
Yptb5(pSMV4) and Yptb5(pSMV8) in mice had similar patterns. Mice vaccinated orally with 5
108
Yersinia pseudotuberculosis
Vaccine
CFU of either Yptb5(pSMV4) or Yptb5(pSMV8) strain were primed high antibody titers with a balanced
Plague Th1/Th2 response, also developed potent T-cell responses with significant productions of IFN-c, IL-17A
And regulated delayed attenuation and TNF-a. Immunization with each mutant strain conferred complete protection against pulmonary
challenge with 5.5
103 CFU (55 LD50) of Y. pestis, but partial protection (50% survival) against 100
LD50 of Y. pestis. Our results demonstrate that arabinose-dependent regulated delayed fur shut-off is an
effective strategy to develop live attenuated bacterial vaccines while retaining strong immunogenicity.
Ó 2020 Elsevier Ltd. All rights reserved.

1. Introduction play features of the wild-type virulent pathogen at the time of

Plague is one of the most feared infectious diseases caused by
Yersinia pestis. Three well-known plague epidemics (Justinian,
Black death and Modern plague) caused a huge historical disaster
and claimed more than 200 million human lives [1,2]. Currently,
no FDA-approved vaccine is available yet. Pneumonic plague
caused by pulmonary infection of Y. pestis is the most lethal form
[1]. So, development of a safe and effective vaccine for human
application is a priority, particularly for people lives in plague
endemic regions. Live vaccines established on Y. pseudotuberculo-
sis, the Y. pestis progenitor [3], showed induction of potent
antibody- and cell-mediated responses in mice, and provided sig-
nificant protection against pulmonary challenge with virulent Y.
pestis strains [4–7]. The basis built on previous studies can allow
us seeking different means to optimize the live attenuated Y.
pseudotuberculosis vaccine.
A regulated delayed attenuation system was developed in Sal-
monella [8] and Yersinia [6,9], in which the recombinant strains dis-
⇑ Corresponding author.
E-mail address: sunw@amc.edu (W. Sun).
immunization to enable strain to effectively colonize lymphoid tis-
sues and then become completely attenuated in vivo to preclude
inducing disease symptoms. Studies demonstrated that antibodies
against iron regulated outer membrane proteins (IROMPs), which
block essential iron uptake of the bacterial pathogens, were cap-
able of cross-reacting with IROMPs synthesized by other pathogens
belonging to the same species [10]. In S. Typhimurium, deletion of
fur encoding ferric uptake regulator enabled constitutive expres-
sion of IROMPs, attenuating infection of orally-challenged mice
by 2–3 log fold [11]. To achieve a high constitutive level of synthe-
sis of all components for iron acquisition including IROMPs, a S.
Typhimurium strain with a DPfur::TT araC PBAD fur deletion-
insertion mutation displayed that expression of the fur gene was
solely dependent on the presence of arabinose, instead of iron con-
centration [8]. Growth of the S. Typhimurium mutant strain in
media with a low level of arabinose resulted in very good coloniza-
tion of lymphoid tissues, high attenuation and very high levels of
induced protective immunity [8]. Yersinia iron acquisition systems
are required for full virulence [12–16]. Both ybt and yfe loci in Y.
pestis and Y. pseudotuberculosis have been found to be 97–100%
identical. The Yersinia Fur protein represses transcription of genes
located at ybt and yfe loci in the presence of excess iron [17,18].

https://doi.org/10.1016/j.vaccine.2020.03.055
0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.
 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728 3721

Yersinia grown in the iron deprived media results in enhanced
synthesis of major IROMPs such as Psn, Irp1, Irp2, YbtA and so on
[19–21]. Thus, a live attenuated Y. pseudotuberculosis strain with
DPfur::TT araC PBAD fur deletion-insertion mutation would enhance
synthesis and exposure of IROMPs in vivo, predictably priming
robust immune responses and enhancing attenuation.
Previous findings have demonstrated a truncated FliC180 in Sal-
monella deletes the 180 amino acids containing the antigenically
hypervariable domain of the flagellin antigen, but retains the con-
served N- and C-terminal regions that interact with Toll like
receptor-5 (TLR5) on immune cell surface to recruit/stimulate
innate immune responses [22,23]. Immunization with flagellin
mixed or fused with Y. pestis antigen conferred clearly protection
against plague challenge in mice and nonhuman primates [24–26].
Based on above information, we incorporated DPfur::TT araC
PBAD fur deletion-insertion mutation into a live attenuated Y. pseu-
dotuberculosis construction v10069 (Dasd DyopJ DyopK) [4] to
construct a novel vaccine strain, Yptb5 (Dasd DyopJ DyopK DPfur::
TT araC PBAD fur). Furthermore, we fused FliC180 from S. Typhimur-
ium with Y. pestis LcrV antigen and delivered this fusion antigen by
Yptb5 strain to enhance immunogenicity. Mouse studies demon-
strated oral immunization with Yptb5 delivering the FliC180-
LcrV fusion antigen induced Y. pestis specific humoral and cell
mediated immunity and conferred significant protection against
pneumonic plague.
2. Materials and methods
2.1. Bacterial cultures and media
Bacteria and plasmids used in this study were listed in Table 1.
Escherichia coli v7213 as a suicide plasmid donor strain was grown
routinely at 37 °C in Luria Bertani (LB) broth or on LB agar plates
supplemented with diaminopimelic acid (50 lg/ml, DAP) or chlo-
ramphenicol (25 lg/ml, Cm) as necessary. The Y. pseudotuberculo-
sis strain used in this study was routinely grown in LB medium at
28 °C. LB plates containing 5% sucrose were used for sacB gene-
based counter-selection in allelic exchange experiments for
mutant constructions. Yersinia selective agar supplemented with
Cefsulodin-Irgasan-Novobiocin (CIN) was applied for bacterial
enumeration in different organs of mice administrated with Y.
pseudotuberculosis. Y. pestis KIM6+(pCD1Ap) was used for chal-
lenge studies as previously reported [4] and grown routinely in
heart-infusion broth (HIB) or HIB- Congo red agar plates at 28 °C.
HIB-Congo red agar plates were used to confirm the pigmentation
(Pgm+) phenotype of virulent Y. pestis strain [27].
2.2. Plasmid and strain constructions
All primers used in this study were listed in Supplemental infor-
mation Table S1. A primer set of FliC180-F/FliC180-R was used for
amplifying fliC180 DNA fragment encoding truncated FliC180 from
S. Typhimurium UK-1 mutant strain, v9206 (received from Dr. Roy
Curtiss). Another primer set of LcrV-F/LcrV-R was used to amplify
lcrV DNA fragment of Y. pestis. The chimeric DNA fragments
(fliC180-lcrV) were fused by overlapping PCR using a primer set
of FliC180-F/LcrV-R. The fliC180-lcrV fragment digested by NcoI
and HindIII was cloned into the same sites of two Asd+ plasmids,
pYA3332 (p15A ori) and pYA3337 (pSC101 ori) to generate pSMV4
and pSMV8, respectively (Table 1 and Fig. 2A). Primer sets Pfur-1/
Pfur-2 and Pfur-3/Pfur-4 were used for amplifying fur containing
its original SD sequence (SD-fur), and YPTS_11830 (113 to
760 bp upstream of fur) fragments from wild-type Y. pseudotu-
berculosis PB1+strain, respectively. The SD-fur and YPTS_11830
fragments were cloned into the XhoI/SacI sites and PstI/SphI sites
of pYA3700, respectively to form pSMV31. The DPfur::TT araC PBAD
fur DNA fragment was digested from plasmid pSMV31 by SacI/SphI,
blunted by T4 DNA polymerase and ligated into pRE112 to gener-
ate a suicide plasmid pSMV32. All constructed plasmids were con-
firmed by DNA sequencing.
The procedures for constructing the Y. pseudotuberculosis
mutant were described as previous studies [4,6]. Briefly, the sui-
cide plasmid pSMV32 (DPfur::TT araC PBAD fur) was conjugationally
transferred from E. coli v7213 [28] to v10069 (Dasd DyopJ DyopK)
[4]. Single-crossover insertion strains were isolated on TBA agar
plates containing Cm. Loss of the suicide vector after the second
recombination between homologous regions (i.e., allelic exchange)
was selected by using the sacB-based sucrose sensitivity counter-
selection system. The colonies were screened for Cms (chloram-
phenicol sensitive) and verified by PCR using primers Pfur-1/
Pfur-4.
2.3. In-vitro assay of TLR5 activities
To determine stimulatory activity of FliC180-LcrV fusion via
TLR5, HEK-BlueTM-mTLR5 cells (InvivoGen, CA, USA) were main-
tained at 37 °C with 5% CO2 in DMEM (Gibco BRL, Grand Island,
NY, USA) containing 10% FBS supplemented with 100 lg/ml

Table 1
Strains and plasmids used in this study.

Strain or Plasmid Genotype or relevant characteristics Source

Strains
E. coli v6212 F– k– /80 D(lacZYA-argF) endA1 recA1 hsdR17 deoR thi-1 glnV44 gyrA96 relA1 DasdA4 [7]
E. coli v7213 thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 DasdA4 recA1 RP4 2-Tc::Mu [kpir]; Kmr [7]
Salmonella Typhimurium fliC180 Dr. Roy Curtiss
UK-1 mutant v9206
Y. pseudotuberculosis PB1+ Serotype O:1b Dr. Roy Curtiss
Y. pseudotuberculosis v10069 Dasd206 DyopJ315 DyopK108 [4]
Yptb-A5 Dasd206 DyopJ315 DyopK108 DPfur::TT araC PBAD fur This study
Y. pestis KIM6+(pCD1Ap) Pgm+, pMT1, pPCP1, pCD1Ap [27]
Plasmids
pRE112 Suicide vector, Cmr, mob- (RP4)R6K ori, sacB [6]
pYA3332 Asd+, p15A ori [6]
pYA3337 Asd+, pSC101 ori [43]
pYA4505 The fliC180 gene fragment under Ptrc promoter in an Asd+ plasmid (pBR ori) Dr. Roy Curtiss
pSMV4 The fliC180-lcrV DNA fragment was cloned into pYA3332 This study
pSMV7 The lcrV gene fragment was cloned into pYA3332 This study
pSMV8 The fliC180-lcrV DNA fragment was cloned into pYA3337 This study
pSMV31 The SD-fur and YPTS_11830 fragments cloned into the XhoI/SacI sites and PstI/SphI sites of pYA3700 This study
pSMV32 The DPfur::TT araC PBAD fur DNA fragment was cloned into pRE112 This study
3722 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728
penicillin, 100 lg/ml streptomycin and 100 mg/ml Normocin. Cells
were seeded at a density of 5
104 cells per well in 96-well tissue
culture plates (Costar, Washington, DC) and were stimulated with
bacterial lysate from different strains (final concentration 20
lg/ml) for 6 h. HEK-BlueTM-Null cell without TLR5 was used as
experimental control. Relative NF-jB activity was determined by
measuring the embryonic alkaline phosphatase (SEAP) activity
secreted into culture media according to the manufacturer’s
instructions.
2.4. Mice and ethical statement
Animal care and experimental protocols were in accordance
with the NIH ‘‘Guide for the Care and Use of the laboratory Ani-
mals” and were approved by the Institutional Animal Care and
Use Committee at Albany Medical College (IACUC protocol# 20-
01001). Six-week-old, male and female Swiss Webster mice were
purchased from Charles River Laboratories (Wilmington, MA). Mice
were acclimated for one week before experiments.
2.5. Bacterial inoculation and immunization
The overnight grown cultures of Yptb5(pSMV4) and Yptb5
(pSMV8) were re-inoculated in fresh LB broth with arabinose
(0.05% v/v), respectively. Bacterial cultures were grown at 28 °C
with 180 rpm constant agitation until exponential phase (O.
D.600nm 0.8–0.9). Bacterial cells were pelleted at 4000
g for
12 min and resuspended in sterile phosphate buffered saline
(PBS) pH 7.4 with required volume. Groups of mice (10 mice each
group) were deprived of food and water for 6 h to minimize intesti-
nal contents and gavaged 0.2 ml bacterial suspension to each
mouse with a single dose of 5.5
108 colony forming units
(CFU) by using 20 gauge feeding needle. Equal number of sham
mice were given 0.2 ml of sterile PBS [4,7].
2.6. Colonization of Y. pseudotuberculosis mutant
9 deviation (SD) obtained from minimum 3 biological replicated and
Groups of mice were gavaged with 5.5
10 CFU of Y. pseudo-
tuberculosis to assess the kinetics of bacterial burden in different
vital organs including intestine, spleen, liver and lung. At day 3, 6
and 9 post infection, 3 mice from each group were humanly euth-
anized and perfused with 5 ml sterile PBS before extracting lung,
liver, spleen and Peyer’s patch, aseptically. Tissue samples were
homogenized by bullet blender (Bullet Blender Blue; Averill Park,
NY, USA) and serially diluted homogenates were spread on Yersinia
selective agar plates (CIN agar) in duplicates. After 48 h incubation
at 28 °C, CFU were counted and calculated according to the initial
weight (mg) or/and volume (ml) of the organ [4,7].
2.7. Antibody responses
On day 14 and 28 post immunization, blood was collected from
immunized and sham mice by sub-mandibular vein puncture for
antibody analysis. For measuring secreted IgA in bronchoalveolar
lavage fluid (BALF), 4 representative mice from each group were
euthanized at 42nd day post immunization and BALF was collected
as previous description [4]. An enzyme-linked immunosorbent
assay (ELISA) was used to measure antibodies in serum and BALF
to YpL (Y. pestis whole-cell lysate) or LcrV [4].
2.8. Antigen specific T-cell responses
Single cells obtained from spleens and collagenase treated lungs
were seeded (2
106) in 12-well cell culture plates. Cells were in-
vitro stimulated with 20 lg/ml YPL or LcrV protein for 72 h at 37 °C
and 5% CO2. Four hours before collection, cell culture media in each
well were supplemented with brefeldin-A and monensin cocktail
(1:1 ratio) to plug Golgi-mediated cytokine secretion. For flowcyto-
metric analysis of T-cell population and their corresponding
cytokines, induced cells were harvested and re-suspended in FACS
staining buffer containing CD16/32 antibodies (1:200) for 10 min
on ice. T-cell specific markers were stained using anti-mouse
CD3 (FITC), CD4 (PE) and CD8 (APC) antibodies followed by intra-
cellular cytokines (IFN-c, Percp Cy5.5; TNF-a, HV510; IL-2, PECy7;
IL17A, APCCy7) staining by using BioLegend Perm-fix solution and
buffer as per manufacturer’s protocol. All antibodies were pur-
chased from BioLegend. The entire staining process was performed
on ice with 1 h incubation at each step. Events were acquired on
BD flow cytometers (LSRII) with FACS Diva software and analyzed
by using FlowJo v.10.
2.9. Animal survival study
At 42nd day of immunization, immunized and sham mice were
shifted to ABSL-3 to perform pneumonic plague challenge. Liquid
cultures of Y. pestis KIM6+(pCD1Ap) were grown in HIB supple-
mented with Xylose (0.2% w/v) and ampicillin (100 lg/ml) at
26 °C for overnight. The bacterial cultures were then diluted to
an OD600 of 0.1 in 10 ml fresh HIB with Xylose (0.2% w/v), ampi-
cillin (100 lg/ml), and CaCl2 (2.5 mM) and grown at 37 °C with
constant shaking until 0.6–0.8 of OD600. Bacterial cells were har-
vested by 4000 rpm centrifugation for 12 min and pellet resus-
pended in 1 ml of sterile PBS. Then, the resuspended bacterial
solution was diluted to an appropriate concertation for animal
challenge. Ketamine (xylazine/ketamine 1:5 ratio) anesthetized
mice were infected with 40 ll Y. pestis suspension via nostril
[4,7]. Mortality and morbidity of infected mice were observed daily
for the next 15 days.
2.10. Statistical analysis
All experimental data were presented in mean value ± standard
repeated 2 times in similar condition. Data were analyzed using
GraphPad PRISM 8.0 software. Statistical analyses of data were
evaluated by Two-way ANOVA using Tukey’s post hoc tests. The
log-rank test was used for analysis of the survival curves. Data
represented significance at * P < 0.05, ** P < 0.01, *** P < 0.001,
**** P < 0.0001.
3. Results
3.1. Construction of a regulated delayed fur expression strain, and
determination of FliC180-LcrV synthesis and TLR5 activation
The DPfur::TT araC PBAD fur deletion-insertion mutation was
added on top of v10069 (Dasd DyopJ DyopK) to construct a new
mutant strain designated as Yptb5 (Fig. 1A and Table 1). In Yptb5
mutant strain, fur expression is dependent on the presence of ara-
binose. No Fur was detected in Yptb5 grown in the absence of ara-
binose, while Yptb5 synthesized roughly the same amount of Fur
as wild-type Y. pseudotuberculosis (Fig. 1B). Yptb5 grown in the
absence of arabinose or iron-deprived media significantly
increased levels of Psn (pesticin receptor) (Fig. 1B), an Y. pestis pro-
tective antigen [29,30]. Strain Yptb5 without arabinose supple-
mentation grew slowly at initial and log phases but could reach
the same final OD600 at stationary phase as Yptb5 with arabinose
and its patent strain v10069 at 28 °C in HIB medium (Fig. 1C).
Then, we individually introduced low-copy-number Asd + plasmids,
pSMV4 (p15A ori) and pSMV8 (pSC101 ori) (Fig. 1D) into Yptb5
strain to determine fliC180-lcrV expression. Results showed that
 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728 3723

A B C χ10069
kDa 1.5 Y ptb5
M
0.05% Arabinose - - + Yptb5 +0.05% arabinose
20
Fur
17 1.0
16.7 kDa
11

OD600
0.5
0.05% Arabinose
- - - + +
100μM 2,2’-dipyridyl - + - - +
100 0.0
Psn 0 8 16 24 32 40 48 56
75
55 hours

D E F

**** Yptb5(pYA3332)
2.0

HEK-blue TLR5 stimulation

**** Yptb5(pYA4505)
kDa M Yptb5(pSMV4)
1.5

O.D. (655nm)
Yptb5(pSMV7)
100 FliC180-LcrV **** Yptb5(pSMV8)
70
1.0 ****
55 71.3 kDa

35 0.5
25
0.0
15 HEK-TLR5 HEK-Null

Fig. 1. The features of Y. pseudotuberculosis construction. (A) Schematic chromosome structure of Y. pseudotuberculosis mutants, v10069 (Dasd DyopJ DyopK), and Yptb5
(Dasd DyopJ DyopK DPfur::TT araC PBAD fur). (B) The fur expression in v10069 and Yptb5 cultured in LB media with or without 0.05% arabinose at 28 °C. The psn expression in
v10069 and Yptb5 cultured in LB media with or without 0.05% arabinose and/or 100 lM 2,20 -dipyridyl at 28 °C. (C) The growth curve of v10069 and Yptb5 cultured in LB
media with or without 0.05% arabinose at 28 °C. (D) Physic maps of plasmids pSMV4 (p15A ori) and pSMV8 (pSC101 ori) harboring the fliC180-lcrV fusion gene under Ptrc
promoter. (E) Determine FliC180-LcrV synthesis in Yptb5(pSMV4) and Yptb5(pSMV8). (F) Comparison of the secreted embryonic alkaline phosphatase (SEAP) activities in
HEK-BlueTM cells with or without murine toll-like receptor 5 (TLR5). HEK-BlueTM mTLR5 (InvivoGen) cells were co-cultured with 20 mg/ml whole-cell lysate from Yptb5
(pYA3332), Yptb5(pAY4505, fliC180), Yptb5(pSMV4, fliC180-lcrV), Yptb5(pSMV7, lcrV) and Yptb5(pSMV8, fliC180-lcrV) strains for 6 h, respectively. HEK-BlueTM Null1-v cells or
PBS were used as negative controls. Each symbol represented data obtained from individual well, with mean value bars ± SD. ****, P < 0.0001 was obtained by two-way ANOVA
with Tukey’s post-hoc tests.

the amount of FliC180-LcrV fusion was similar in both Yptb5
(pSMV4) and Yptb5(pSMV8) strains (Fig. 1E).
To determine TLR5-activating abilities of FliC180-LcrV fusion
protein, HEK-blue mTLR5 reported cells containing an NF-jB-
inducible SEAP reporter gene were incubated with 20 lg/ml of sol-
uble protein fraction obtained from whole-cell lysates of each
strain for 6 h. Protein fractions from both Yptb5(pSMV4) and Ypt-
b5(pSMV8) strains synthesizing FliC180-LcrV strongly activated
TLR5 signaling and showed significant increase of SEAP activity
(4–5 fold), although the SEAP activity was lower than that induced
by protein fraction from Yptb5(pYA4505) synthesizing FliC180
alone as a positive control (Fig. 1F). In contrast, protein fractions
from Yptb5(pSMV7) expressing LcrV alone and Yptb5(pYA3332)
harboring an empty plasmid did not show any induction. HEK-
blue null cells without TLR5 incubated with each protein fraction
also did not show any TLR5 stimulation (Fig. 1F). Thus, results indi-
cated that FliC180-LcrV fusion antigen still retained strong activa-
tion of TLR5.
3.2. Kinetics of Y. pseudotuberculosis mutant strains after oral
administration
infection (Fig. 2A). However, the numbers of Yptb5(pSMV4) and
Yptb5(pSMV8) strains in Peyer’s patches gradually decreased and
were substantially lower than that of wild-type PB1+ strain at
day 3, 6 and 9 post-infection and the bacterial titers of Yptb5
(pSMV8) reduced further than those of Yptb5(pSMV4) at day 6
and 9 post administration (Fig. 2A). The PB1+ strain also effectively
colonized in livers and spleens at 3 and steadily heightened, at
6 days and 9 days post-infection (Fig. 2B and C). Interestingly, Ypt-
b5(pSMV8) rapidly disseminated into spleens and livers at 3 days
post-infection compared with Yptb5(pSMV4) and WT PB1+. Then,
titers of both Yptb5(pSMV4) and Yptb5(pSMV8) dramatically
decreased at day 6 and 9 post infection (Fig. 2B and C). Significantly
reduced burdens of Yptb5(pSMV4) and Yptb5(pSMV8) in Peyer’s
patches, spleens and livers clearly manifested their attenuation
properties. The lung burden profiles of Yptb5(pSMV4) and Yptb5
(pSMV8) were also very intriguing. Yptb5(pSMV4) rapidly dissem-
inated to lung in majority of mice at early stages of infection (day
3), while Yptb5(pSMV8) could reach to lung at day 6 post infection,
and both strains were persistent until day 9 post administration.
However, wild-type PB1+ strain was able to reach lungs of half
mice only by day 9 post infection (Fig. 2D).

In order to investigate colonization of mutant strains, Swiss
Webster mice (n = 3 per time point) were gavaged with 200 ll
(5.5
109 CFU) of Yptb5(pSMV4), Yptb5(pSMV8) and wild-type
Y. pseudotuberculosis PB1+. At day 3 post infection, the wild-type
PB1+ infected mice were found with overwhelming infection in
Peyer’s patches and obvious micro-damage in intestinal epithe-
lium, and bacterial load increased progressively at day 6 and 9 post
3.3. Antigen-specific antibody titers in serum and BALF
Sera and BALF obtained from Yptb5(pSMV4) and Yptb5(pSMV8)
immunized mice were evaluated for antibody titers and isotypes
by ELISA using YPL (Y. pestis whole-cell lysate) and LcrV as target
antigens. Mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8)
mounted high anti-YPL IgG titers with similar log10 mean values
3724 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728

A Peyer's patch B spleen

10 **** 10

CFU per gram tissue (log10)

****

CFU per gram tissue (log10 )

**** *** ***
8 **** **** 8
* ***
**
6 6 ***
* ns
4 4

2 2

0 0
day3 day6 day9 day3 day6 day9

PB1+ Yptb5(pSMV4) Yptb5(pSMV8) PB1+ Yptb5(pSMV4) Yptb5(pSMV8)

C liver D lung
10
CFU per gram tissue (log10 )

10

CFU per gram tissue (log1 0 )

8
8
**** **** ****
6 * 6 ***
*
4 4

2 2

0 0
day3 day6 day9 day3 day6 day9

PB1+ Yptb5(pSMV4) Yptb5(pSMV8) PB1+ Yptb5(pSMV4) Yptb5(pSMV8)

Fig. 2. Kinetics of bacterial burden in mice administrated orally with 109 CFU of each Y. pseudotuberculosis PB1+, Yptb5(pSMV4), and Yptb5(pSMV8). Bacterial titers in (A)
Peyer’s patches, (B) spleens, (C) livers and (D) lungs of infected mice at days 3, 6 and 9 post infection. Experiment was performed twice with equal numbers of animals (3 mice
in each group); graphs represented pooled results. Statistical significance among groups were analyzed by two-way ANOVA multivariant applying Tukey’s post-hoc test.
Mean value bars ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.

in sera at 14th day post immunization and maintained high IgG 3.4. T-cell Mediated immune responses
titers without substantial increase at day 28 (Fig. 3A). Analysis of
IgG subclasses (IgG1, IgG2a and IgG2b) to YpL showed that ratios In addition to induction of antibody-mediated humoral immu-
of IgG2a/IgG1 and IgG2b/IgG1 in mice immunized with either Ypt- nity, an increasing body of evidence has demonstrated that an ideal
b5(pSMV4) or Yptb5(pSMV8) were close to 1, respectively (Fig. 3B pneumonic plague vaccine is required to induce cellular immunity
and C). Also, mice immunized with Yptb5(pSMV4) or Yptb5 [31,32]. Therefore, lung and splenic T-cell responses and their cor-
(pSMV8) were primed high anti-LcrV IgG titers with similar log10 responding cytokine productions were analyzed to verify the
mean values in sera at 14th day post immunization (Fig. 3D). At development of cell-mediated protective immunity at mucosal
day 28 post immunization, anti-LcrV IgG titers in the Yptb5 and systemic sites. Lung cells from both Yptb5(pSMV4)- and
(pSMV4)-immunized group were substantial increase, while in Yptb5(pSMV8)-immunized mice in vitro stimulated with YPL
the Yptb5(pSMV8)-immunized group were maintained (Fig. 3D). showed a substantial induction of (CD3+) T-cell populations com-
Ratios of IgG2a/IgG1 and IgG2b/IgG1 to LcrV in mice immunized pared with lung cells from sham mice (Fig. 5A). Higher prolifera-
with either Yptb5(pSMV4) or Yptb5(pSMV8) were also close to 1 tion of lung CD4+ and CD8+ T cells in the Yptb5(pSMV4)-
at day 28 post immunization, respectively (Fig. 3E and F), indicat- immunized group (CD4, 5.5
105) than that in the Yptb5
ing that mixed Th1/Th2 responses were stimulated by oral immu- (pSMV8)-immunized group. In vitro induction by YPL resulted in
nization with both vaccine candidates. higher production of IFN-c in both CD4+ and CD8+ T cells from
Further analysis of IgM and IgA indicated that both immunized Yptb5(pSMV4)-immunized mice than that from Yptb5(pSMV8)-
groups were primed a moderate induction of anti-YPL and -LcrV immunized mice (Fig. 5A). Compared with lung lymphocytes from
serum IgM and IgA titers at day 14 and maintained until day 28 sham mice, incubation of lung lymphocytes with YPL led to sub-
(Fig. S1A, B, C and D). The mucosal immune response at lung was stantial induction of IL-2, IL-17A and TNF-a in CD4+ T-cell subsets
characterized by assessing secretory IgA (sIgA) in BALF obtained of Yptb5(pSMV4)- and Yptb5(pSMV8)-immunized mice, rather in
at the 42nd day post immunization. Mice immunized with Yptb5 CD8+ T-cell subsets (Fig. 5A).
(pSMV4) or Yptb5(pSMV8) produced high levels of YPL-specific Also, substantial T cells (CD3+), and CD4+ and CD8+ T-cell subsets
sIgA titers with similar log10 values, while immunization with Ypt- were proliferated in splenocytes from both Yptb5(pSMV4)- and
b5(pSMV8) generated significantly higher titers of anti-LcrV sIgA in Yptb5(pSMV8)-immunized mice in vitro stimulated with YPL com-
lung than that with Yptb5(pSMV4) (Fig. S1E). No conspicuous anti- pared with cells from sham mice (Fig. 5B), implying development
body titers or isotypes were observed in sera or BALF from PBS of Y. pestis specific cell-mediated systemic immunity. CD4+ T-cell
inoculated mice. subsets in splenocytes from both Yptb5(pSMV4)- and Yptb5
 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728 3725

5.0
A B C
Anti-YPL IgG titers (log10)
4.0 * 2.0 2.0

Anti-YPL IgG2b:IgG1 ration

Anti-YPL IgG2a:IgG1 ration
1.5 1.5
3.0

1.0 1.0
2.0

0.5 0.5
1.0
0.0 0.0

14

28

14

28
0.0

y
day14 day28

da

da

da

da
Yptb5(pSMV4) Yptb5(pSMV8) Yptb5(pSMV4) Yptb5(pSMV8) Yptb5(pSMV4) Yptb5(pSMV8)
Anti-LcrV Serum IgG titers (Log10 )

D 5.0
E F 1.5

Anti-LcrV IgG2b:IgG1 ratio

1.5
Anti-LcrV IgG2a:IgG1 ratio
**** ****
4.0
1.0
1.0
3.0

2.0 0.5
0.5

1.0
0.0
0.0

14

28
14

28

y
0.0

da

da
y

y
da

da

day14 day28
Yptb5(pSMV4) Yptb5(pSMV8)
Yptb5(pSMV4) Yptb5(pSMV8) Yptb5(pSMV4) Yptb5(pSMV8)

Fig. 3. Antibody responses in sera of mice immunized orally with Yptb5(pSMV4), and Yptb5(pSMV8) at day 14 and 28 post immunization, respectively. Y. pestis whole-cell
lysate (YPL) and recombinant LcrV were used as the coating antigens. (A) Serum anti-YPL IgG titers in immunized mice. (B) Ratio of serum anti-YPL IgG2a and IgG1 titers in
mice. (C) Ratio of serum anti-YPL IgG2b and IgG1 titers in mice. (D) Serum anti-LcrV IgG titers in immunized mice. (B) Ratio of serum anti-LcrV IgG2a and IgG1 titers in mice.
(C) Ratio of serum anti-LcrV IgG2b and IgG1 titers in mice. Statistical significance among groups were analyzed by two-way ANOVA multivariant applying Tukey’s post-hoc
test. *, P < 0.05; ****, P < 0.0001. Mean value bars ± SD.

(pSMV8)-immunized mice produced higher levels of IFN-c, IL-2, IL- shown substantial protection against pneumonic plague [4]. How-
17A and TNF-a in response to re-stimulation with YPL than cells ever, histopathological analysis of lung showed moderate sign of
from sham mice (Fig. 5B). Compared with sham mice, CD8+ T-cell infection in v10069 immunized mice, have raised certain safety
subsets in splenocytes from both Ypt5(pSMV4)- and Ypt5 concerns. Since v10069 strain was shown high attenuation in mice
(pSMV8)-immunized mice produced substantial levels of IFN-c, [4], adding any essential gene deletions on top of v10069 leaded to
but displayed limited production of IL-2, IL-17A and TNF-a (Fig. 5B). hyper-attenuation and subsequent loss of immunogenicity during
screening Y. pseudotuberculosis vaccine candidates (unpublished
3.5. Protection against pneumonic plague challenge data). Thus, Yptb5 incorporated with the arabinose regulated
expression of fur gene to enable strain to colonize lymphoid tissues
Groups of mice (10 mice per group, 5 males and 5 females) were as its parent strain v10069 and then become more attenuation
orally immunized with a single dose of each mutant strain (5
108 than v10069 in vivo to preclude inducing disease symptoms,
CFU), with PBS as a negative control. At 42nd day post immunization which would retain immunogenicity but further reduce pathologic
(Fig. 5A), mice immunized with Yptb5(pSMV4) or Yptb5(pSMV8) changes in tissues.
were afforded 100% protection against intranasal challenge with Flagellin, being a potent activator of innate and adaptive immu-
55 LD50 (5.5
103 CFU) of virulent Y. pestis (Fig. 5B), while ~50% sur- nity, was utilized in previous plague vaccine studies [26,33].
vival against intranasal challenge with high dose (100 LD50) of Y. pes- Hence, incorporation of an adjuvant to enhance immune responses
tis (Fig. 5C). At the 14th day post infection, all surviving animals were would be a reasonable approach to maximize protection against Y.
found to have healthy conditions without any external signs of dis- pestis infection. We initially tried to clone the fliC180-lcrV fusion
ease. No bacteria were detected in lungs, livers and spleens of surviv- gene fragment into an Asd+ plasmid with high copy number,
ing animals, thus demonstrating induction of sterilizing immunity. pYA3342 (pBR ori) [34]. However, significantly slower growth of
All sham mice administered with PBS succumbed to infection within Yptb5 harboring this plasmid was observed, which may be due
3–5 days. Male and female immunized mice were found unbiased to overexpression of fliC180-lcrV causing a metabolic burden in
for protection against Y. pestis infection. host strain. Thus, a shift to a low copy plasmid pSMV4 (p15A ori)
or pSMV8 (pSC101 ori) was performed in this study to eliminate
4. Discussion this problem. Yptb5 strain harboring either pSMV4 or pSMV8
showed similar growth rate as Yptb5 harboring an empty plasmid
Our previously designed an Y. pseudotuberculosis vaccine candi- pYA3332 (unpublished data).
date, v10069 (Dasd DyopJ DyopK) harboring plasmid pYA5199 for Our study showed that the titers of Yptb5(pSMV4) and Yptb5
delivering YopEnt138-LcrV by Type three secretion system, was (pSMV8) were significantly lower, but persistence in Peyer’s
3726 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728

patches compared with WT PB1+ strain (Fig. 2A), which was in decreased at day 6 and 9 post inoculation (Fig. 2B and C), which
accordance with a previous report that the highly attenuated yopK might be related with the arabinose-dependent regulated fur
mutant Y. pseudotuberculosis was able to colonize in Peyer’s expression in Yptb5(pSMV4) or Yptb5(pSMV8). In those mutant
patches and mesenteric lymph nodes but failed to cause systemic strains, Fur might be completely shut off at day 6 and 9 post inoc-
infection in mice [35]. Somehow, Yptb5(pSMV4) or Yptb5(pSMV4) ulation causing further attenuation. Intriguingly, both Yptb5
could reach spleen and liver more rapidly than WT PB1+ strain at (pSMV4) and Yptb5(pSMV8) could persist higher bacterial titers
day 3 post administration, but titers of mutant strains significantly than WT PB1+ strain in lungs by day 9 post infection (Fig. 2D).

LUNG SPLEEN
A B ****
*** 30.0 150.0
***

T-cell population (x106 )

CD3+ T-cell population (x106)

T-cell population (x105 )

2.5 *** ** ***
6.0 ** ****
T-cell population (x10 5)

2.0 20.0 100.0

4.0
1.5
** ***
10.0 50.0
1.0 *** *
2.0
0.5
0.0 0.0
CD4 CD8
0.0 0.0
CD4 CD8 Yptb5(pSMV4) Yptb5(pSMV8)
sham Yptb5(pSMV4) Yptb5(pSMV8)
Yptb5(pSMV4) Yptb5(pSMV8) sham
sham Y ptb 5(pSMV4 ) Yptb5(p SMV8)
sham

CD4+ T-cell population (x105 )

5
CD8+ T-cell population (x10 )

80.0 15.0

CD8+ T-cell population (x105)

***
CD4+ T-cell population (x105)

2.0
**** ****
4.0 *** ** ****
1.5 60.0
3.0 10.0
*** 40.0
1.0 ****
2.0 ***
** *** *** 5.0
20.0 ***
*** * *** 0.5
1.0

0.0 0.0 0.0

0.0 IFN-γ IL2 IL17A TNF-α IFN-γ IL2 IL17A TNF-α IFN-γ IL2 IL17A TNF-α
IFN-γ IL2 IL17A TNF-α

sham Yptb5(pSMV4) Yptb5(pSMV8) sham Yptb5(pSMV4) Yptb5(pSMV8)

sham Yptb5(pSMV4) Yptb5(pSMV8) sham Yptb5(pSMV4) Yptb5(pSMV8)

Fig. 4. Analysis of antigen-specific T-cell responses from lungs and spleens. After the 42nd day of immunization, lymphocytes were aseptically isolated from lungs and
spleens of mice and in vitro stimulated with Y. pestis whole cell lysate (YPL) at 20 mg/ml for 72 h to assess specific CD4+ and CD8+ T cells producing IFN-c, IL-2, IL-17and TNF-
a. The cells from sham mice were considered as controls. (A) Lung total T cell numbers, numbers of CD4+ and CD8+ T-cell subsets, numbers of CD4+ IFN-c+, IL-2+, IL-17+ or
TNF-a+ positive cells, and numbers of CD8+ IFN-c+, IL-2+, IL-17+ r TNF-a+ positive cells. (B) Spleen total T cell numbers, numbers of CD4+ and CD8+ T-cell subsets, numbers of
CD4+ IFN-c+, IL-2+, IL-17+ or TNF-a+ positive cells, and numbers of CD8+ IFN-c+, IL-2+, IL-17+ or TNF-a+ positive cells. Each symbol represented data obtained from
individual mice, with horizontal mean value bars ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 were obtained by two-way ANOVA with Tukey’s post-hoc tests.

A Day 0 14 28 42 57

Immunization 1st 2nd

by oral gavage bleeding bleeding Challenge End

B 5.5 × 10 3 CFU C 1.04 × 10 4 CFU

100
100
P ercent survival

75
Survival (%)

75
****

50
50
***

25
25

0
0 0 5 10 15
0 3 6 9 12 15
Days post infection
Days post infection
Sham Yptb5(pSMV4) Yptb5(pSMV8)
sham Yptb5(pSMV4) Yptb5(pSMV8)

Fig. 5. Survival of mice challenged by virulent Y. pestis KIM6+(pCD1Ap). Groups of Swiss-Webster mice (n = 10, 5 males and 5 females) were orally vaccinated with a dose of
5
108 CFU of Yptb5(pSMV4), Yptb5(pSMV8), PBS as a negative control. (A) Schematic strategy of the entire animal experiment. (B) Swiss-Webster mice were intranasally
challenged with a medium dose of Y. pestis (5.5
103 CFU). (c) Swiss-Webster mice were intranasally challenged with a high dose of Y. pestis (1.04
104 CFU). Mortality and
morbidity were recorded in surviving mice for 15 days post infection. The log-rank test was used for analysis of the survival curves. (***, P < 0.001; ****, P < 0.0001).
 A.K. Singh et al. / Vaccine 38 (2020) 3720–3728
Regarding previous reports, pathogenic bacteria loss of Fur had
dramatic attenuation by oral administration and lower dissemina-
tion to second lymphoid or non-lymphoid organs [8,36–38]. So,
this discrepancy might be due to alteration of Y. pseudotuberculosis
invasion by fur mutant or different mouse lines used between this
study and other studies, but the exact mechanisms behind those
observations need to be investigated further.
Studies suggested that a plague vaccine candidate primarily
stimulating antibody-mediated humoral immunity was not
enough [25,39,40], but induction of humoral response was able
to mirror cellular response to certain extend. Antibody analysis
showed that Yptb5(pSMV4)- or Yptb5(pSMV8)-immunization
induced a balanced Th1/Th2 response (Fig. 3), which implies con-
current induction of T-cell responses with significant productions
of IFN-c, IL-17A and TNF-a in both lungs and spleens (Fig. 4) and
is consistent to previous studies [40,41]. Both cellular and humoral
immunity were primed by Yptb5(pSMV4) or Yptb5(pSMV8) strain
afforded substantial protection against pulmonary Y. pestis infec-
tion (Fig. 5). In comparison to protective efficacy of v10069
(pYA5199) [4], there was no significant difference observed in
Yptb5(pSMV4)- or Yptb5(pSMV8)-immunized mice (Fig. 5). Possi-
ble explanation is that a single-dose immunization of live Y. pseu-
dotuberculosis vaccine candidates may not go beyond the maximal
cap of protection against pneumonic plague. Another possible rea-
son may be that lack of F1 antigen in above vaccine candidates
can’t reach comprehensive protection. Recently, a phase I human
clinical trial showed that the Flagellin-F1/V vaccine exhibited a
dose dependent increase in immunogenicity and induced both
humoral and cellular immune responses [33,42]. So, we are going
to incorporate another important Y. pestis antigen F1 into this
new Y. pseudotuberculosis construction, as well as alter immuniza-
tion regime using homologous or heterologous prime-boost strat-
egy to further enhance protection.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgement
We thank Drs. Roy Curtiss III and Shifeng Wang at the Univer-
sity of Florida for sharing different strains and pYA plasmids with
us. This work was supported by the National Institutes of Health
grant R01AI125623 and R21AI139703 to WS and the Albany
Medical College start-up fund.
Contributions
Conceived and designed the experiments: WS and AKS. Per-
formed the experiments: AKS, XRW and WS. Analyzed the data:
AKS and WS. Wrote the paper: AKS and WS.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.vaccine.2020.03.055.
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