Vaccine 34 (2016) 5762–5767

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

Characterization of the protective immune response to Yersinia

pseudotuberculosis infection in mice vaccinated with an LcrV-secreting
strain of Lactococcus lactis
Catherine Daniel 1, Marie Titecat 1, Sabine Poiret, Delphine Cayet, Denise Boutillier, Michel Simonet,
Jean-Claude Sirard, Nadine Lemaître, Florent Sebbane ⇑
Univ. Lille, Inserm, CNRS, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL – Center for Infection and Immunity of Lille, F-59000 Lille, France

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

Article history: Background: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudo-
Received 10 May 2016 tuberculosis and is considered to be a significant problem in veterinary medicine. We previously found
Received in revised form 29 September that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium
2016
response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseu-
Accepted 30 September 2016
Available online 11 October 2016
dotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited
protective response and at determining the duration of vaccine-induced immunity.
Methods: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were
Keywords:
Yersinia
immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of
LcrV Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+
Vaccination or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the num-
Lactococcus lactis ber of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intra-
venous injection of Y. pseudotuberculosis was followed up to 9-months.
Results: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by
Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immu-
nized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that
activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of
LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in
the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV
decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge
at 9 months post-vaccination.
Conclusion: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective
immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction present, the PseudovacÒ vaccine (a mixture of killed strains of Y.

The Gram-negative pathogen Yersinia pseudotuberculosis causes
outbreaks of pseudotuberculosis in livestock and in animals kept in
zoological and wildlife parks [1–8]. Hence, this pathogen is a
significant concern in veterinary medicine. Vaccination is the most
effective way of preventing and controlling this zoonosis [9]. At
⇑ Corresponding author at: Equipe Peste et Yersinia pestis, Centre d’Infection et
d’Immunité de Lille, INSERM U1019-CNRS UMR 8204, Institut Pasteur de Lille, 1 rue
du Professeur Calmette, F-59019 Lille cedex, France.
E-mail address: florent.sebbane@inserm.fr (F. Sebbane).
1
Contributed equally to this work.
pseudotuberculosis of various serotypes) is the only vaccine against
pseudotuberculosis, and is currently used in zoos. However,
PseudovacÒ’s efficacy is not well established [10]. A promising
alternative to PseudovacÒ is vaccination based on the LcrV protein
(a 37-kDa protein encoded by a gene on the 70-kb plasmid pYV
harbored by Y. pseudotuberculosis). In fact, LcrV was the first viru-
lence determinant in pathogenic Yersinia to be discovered in the
mid-1950s, and is currently considered to be a major pathogenicity
factor [11]. LcrV is an essential component of a type III secretion
system (T3SS) that exports Yersinia outer proteins (Yops) into the
cytosol of phagocytes, where they disrupt cell functions involved
in immune responses and activate anti-inflammatory responses

http://dx.doi.org/10.1016/j.vaccine.2016.09.060
0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.
 C. Daniel et al. / Vaccine 34 (2016) 5762–5767
[12,13]. The LcrV protein is located at the tip of the Yersinia T3SS
‘‘needle”, where it has a crucial role in the formation of a pore in
the eukaryotic cell membrane (through which the Yops are
translocated into the host cell’s cytosol). After pore formation, LcrV
is released in the tissues, where it interacts with cell surface toll-
like receptor 2 receptor on phagocyte. This triggers the production
of the anti-inflammatory cytokine interleukin (IL)-10, which inhi-
bits the secretion of pro-inflammatory cytokines interferon (IFN)-
c and tumor necrosis factor (TNF) [11]. In addition to its role as a
virulence factor and an immunomodulator, LcrV also provides
potent protection against experimental Yersinia infections by stim-
ulating both humoral and cellular immune responses [14]; accord-
ingly, LcrV has been proposed as a potential vaccine component.
Safe microorganisms that (i) deliver bacterial, viral and parasitic
antigens and (ii) elicit mucosal, systemic and cell-mediated
immune responses have the potential to be highly efficacious vac-
cines [15,16]. Several lactic acid bacteria (LAB, such as Lactococcus
lactis, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus
acidophilus) have ‘‘generally recognized as safe” (GRAS) status
and can be administered intranasally to mice. Indeed, LAB vaccine
carriers (primarily L. lactis) have been shown to confer protection
against infectious challenges [17]. With a view to developing a vet-
erinary vaccine against pseudotuberculosis, we previously built a
recombinant Lactococcus lactis strain that secretes the low-
calcium response V (LcrV) antigen from Yersinia pseudotuberculosis
(henceforth referred to as Ll-LcrV) [18]. We showed that (i) intra-
nasal instillation of Ll-LcrV triggered a mixed Th1/Th2 immune
response in mice, and (ii) vaccinated animals survived a normally
lethal dose of Y. pseudotuberculosis administered by the oral route
or by the intravenous route. These findings indicated that the vac-
cination protects against different forms of the disease, including
the most severe (systemic) form. In the present study, we charac-
terized the immunologic basis of the LcrV-elicited protective
response and determined the duration of vaccine-induced immu-
nity in animals developing the most severe form of the disease.
To this end, an intravenous challenge model was used.
2. Materials and methods
2.1. Bacterial strains and growth conditions
L. lactis MG1363 Ll (containing the expression plasmid pNZYR)
and Ll-LcrV (harboring the pNZYR derivative pMEC237, which
bears the PCR-generated Y. pseudotuberculosis lcrV gene [nucleotide
1–984, GenBank accession EU616676] fused to the secretion signal
of the lactococcal Usp45 protein and located downstream of the
lactococcal Usp45 promoter) were used [18]. L. lactis strains were
cultivated at 30 °C in M17 medium (Difco) supplemented with
0.5% glucose and 10 mL 1 chloramphenicol. Y. pseudotuberculosis
IP32777 (pYV-positive) was also used and was grown in Lysogeny
broth (LB) or agar at 28 °C.
2.2. Mice
Seven-week-old female BALB/c mice were purchased from
Charles River Laboratories and housed in animal facilities at the
Institut Pasteur de Lille. The animals were kept in individually ven-
tilated cages and handled in a vertical laminar flow cabinet (class II
A2; Esco). All animal experiments complied with current national
and institutional regulations and ethical guidelines (A59107, Insti-
tut Pasteur de Lille), and animal protocols were approved by the
local investigational review board.
5763
2.3. Immunization
BALB/c mice were immunized intranasally with lactococcal
strains producing (or not) LcrV (Ll-LcrV and Ll, respectively). A dose
of 1  109 bacteria was intranasally administered on days 1, 2, 22,
23, 43 and 44, as described previously [18]. A 10-fold smaller dose
did not induce immunity against pseudotuberculosis (unpublished
data).
2.4. Immunostaining of splenocytes and flow cytometry analysis
Spleens were removed from the mice on day 53 (i.e. 11 days
after the last immunization challenge) and digested with
1 mg mL 1 collagenase IA (Sigma) and 40 lg mL 1 DNase I (Sigma)
for 10 min at 37 °C. After pre-incubation with the 2.4G2 mono-
clonal antibody (mAb), in order to block non-antigen-specific bind-
ing of immunoglobulins to the Fcc receptors CD16/CD32,
splenocytes were stained for 30 min at room temperature with
specific fluorescent antibodies directed against CD3e (mAb 145-
2C11), CD19 (mAb 1D3), CD4 (mAb GK1.5), NKp46 (mAb 29A1.4),
CD3 (mAb 17A2), CD25 (mAb PC61.5), CD8 (mAb 53-6.7), CD45
(mAb 30-F11), TCRb (mAb H57-597), TCRdc (mAb GL3) and CD27
(mAb LG.3A10) purchased variously from Becton Dickinson, BioLe-
gend and eBioscience. The APC-conjugated tetramer specific for
invariant natural killer T (iNKT) cell ligand (PBS-57 glycolipid)-
loaded CD1 tetramer was obtained from the NIAID Tetramer Facil-
ity. Flow cytometry was performed on a BD FACSCanto cell ana-
lyzer and data were analyzed with BD FACSDiva software.
2.5. LcrV-specific antibody titer assay
LcrV-specific IgG levels in sera were determined using an ELISA,
as described elsewhere [18].
2.6. T lymphocyte depletion
Immune-cell-specific mAbs were used for in vivo depletion of
CD4, CD8 and CD25 lymphocyte subsets. Depletion of mouse-
specific T cell populations was performed via two intraperitoneal
injections (24 h apart) of 100 lg of a purified anti-CD4 (clone
GK1.5) [19] or anti-CD8 (clone YTS 169.4, BioXCell), or anti-CD25
(clone PC61) mAb [20]. Control (non-depleted) mice were treated
identically with an isotype mAb (clone ATCC HB 152 or LTF-2 from
BioXCell). The efficacy of T cell depletion was checked by flow
cytometry analysis of splenocytes 36 h after initiation of the mAb
treatment. Depletion was measured to be >93.5%, 99.9%, and
>99.7% with anti-CD4, anti-CD8 and anti-CD25 antibodies,
respectively.
2.7. Serum transfer
Donor blood was collected by cardiac puncture. After blood
clotting, sera were separated by centrifugation and pooled. The
pooled serum was stored at 20 °C until required. Recipient mice
were intravenously infused with 300 lL of serum.
2.8. Mice infection
Experimental infections were performed in a biosafety level 2
facility. The 50% lethal dose for intravenous administration of Y.
pseudotuberculosis IP32777 had previously been determined to be
<102 [18]. Two weeks after the last intranasal immunization with
Ll or Ll-LcrV, groups of 10 animals were challenged with
Y. pseudotuberculosis. Mice received an intravenous infusion of
5764 C. Daniel et al. / Vaccine 34 (2016) 5762–5767

overnight-grown Y. pseudotuberculosis (1  103 CFU in 300 lL of 3. Results and discussion

sterile PBS) and mortality was monitored daily. At day 5 post-
challenge, mice were sacrificed. Spleens and livers were aseptically
harvested and then homogenized with an Ultra Turrax homoge-
nizer (IKA-Werke). Yersinia in spleen and liver homogenates were
counted by plating serial dilutions onto LB agar. Mice were infected
via the intravenous route because we sought to (i) determine the
protective role of LcrV during the most severe form of infection
and (ii) reduce the number of animals for sacrifice and thus limit
inter-individual variability.
2.9. Statistical analysis
Survival rates and time courses in the different groups of
infected mice were compared using a log-rank test. All other data
were analyzed using a one-way analysis of variance or a non-
parametric Mann-Whitney U test. The threshold for statistical
significance was set to p < 0.05.
To characterize the immunological basis of the LcrV-elicited
protective response, in a first step we assessed, by flow cytometry,
the percentage of NK cells, iNKT cells, B and T (CD4+, CD8+, and cd)
lymphocytes relative to total splenocytes in BALB/c mice at day 53
post-intranasal immunization with control Lactococcus (Ll) or Lac-
tococcus expressing LcrV (Ll-LcrV) (Fig. 1). Relative to controls,
counts of splenic NK, iNKT, and cd T cells in Ll-LcrV-immunized
animals were normal; in contrast, the percentage of B lymphocytes
was significantly lower and the percentages of CD4+ and CD8+ T
cells were significantly higher. These data indicate that T and B
lymphocytes (but not non-conventional lymphoid cells) were
affected by Ll-LcrV immunization.
In Ll-LcrV vaccinated mice inoculated intravenously with a nor-
mally lethal dose of Y. pseudotuberculosis, we had previously found
that Y. pseudotuberculosis grows in the spleen and liver during the
first five days post-challenge but is then eliminated over the fol-
lowing two weeks. In contrast, non-vaccinated mice died by day

Fig. 1. Flow cytometry analysis of splenocytes from LcrV+ lactococci-immunized mice. BALB/c mice were in immunized with LcrV+ or with LcrV (mock) L. lactis. Populations
of NK, iNKT, B and T (CD4+, CD8+ and cd) cells in the spleen were analyzed at day 53 post-immunization using flow cytometry. (A) Gating strategy for the identification of
splenocytes. After gating on CD45+, spleen cells were identified as follows: B (CD3negCD19+), T (CD3+CD19neg), CD4 (CD3+CD4+CD8neg), CD8 (CD3+CD4negCD8+), cd (CD3+-
TCRb+), NK (CD3negNKp46+), and NKT (Tetramer+TCRb+). (B) Percentages of spleen cell populations, relative to total spleen cells. Bars represent the mean ± SD from three
individuals. Data are shown for one of two individual experiments. ⁄, p < 0.05 vs. control-vaccinated mice in a non-parametric Mann-Whitney test.
 C. Daniel et al. / Vaccine 34 (2016) 5762–5767 5765

5 or 6 post-challenge [18]. Given the role of T-cells in vaccine-
induced protection, we next sought to evaluate the respective con-
tributions of CD4+ and CD8+ cells in systemic pseudotuberculosis,
the most severe form of infection. To evaluate the contribution of
CD4+ and CD8+ cells to vaccine-induced protection, we depleted
CD4- or CD8-expressing T-cells by administering specific antibod-
ies to immunized mice prior to challenge. We then harvested the
depleted animals’ spleens and livers at day 5 after the lethal chal-
lenge, measured the organs’ bacterial loads and compared these
values with those of Ll-immunized animals treated with an isotype
(control) antibody. The results are presented in Fig. 2. As expected
and as previously described [18], LL-LcrV immunization of mice
was associated with a 1000- to 10,000-fold lower Yersinia load in
the spleen and liver of animals. Furthermore, animals having
received anti-CD4 mAb had significantly higher bacterial counts
in the organs. Animals having received anti-CD8 mAb also had
higher splenic and hepatic bacterial loads than controls, although
the differences were not statistically significant. The same experi-
mental strategy was carried out to establish whether the CD4+ cells
involved in the protection conferred by LL-LcrV were activated or
not. CD25 is strongly expressed by activated T lymphocytes as well
as regulatory T lymphocytes. As shown in Fig. 2, depletion of CD25-
expressing cells resulted in significant higher Yersinia counts in the
livers and spleens of immunized mice. Our results strongly support
that activated CD4+ T lymphocytes are necessary for vaccine-
induced protection, whereas CD8+ T cells may have a moderate
role. However, these data cannot rule out the involvement of reg-
ulatory T cells because the latter also express CD25.
We next sought to better define the role of B cells by establish-
ing whether the adoptive transfer of LcrV-specific antibodies from
LL-LcrV-immunized animals could protect naive mice against a
normally lethal Y. pseudotuberculosis infection. On day 53 post-
immunization, serum collected from LL-LcrV-vaccinated mice
(which contains anti-LcrV antibodies [Fig. 3A]) was intravenously
injected into naive BALB/c mice; 24 h later, animals were intra-
venously challenged with a lethal dose of Y. pseudotuberculosis.
As a control, serum from LL-immunized mice was administered
to naive recipients. Splenic and hepatic Yersinia counts in both
groups of mice were determined at day 5 post-infection (Fig. 3B).
The hepatic bacterial load was 10 times lower (p < 0.05) in BALB/
c mice having received serum from LL-LcrV-vaccinated animals
than in control animals, whereas significant bacterial clearance

Fig. 2. Effect of depleting CD4+-, CD8+- and CD25+-expressing cells on the LcrV-triggered protective immunity against Y. pseudotuberculosis. BALB/c mice were immunized
with LcrV+ or LcrV (mock) L. lactis. At day 53 post-immunization, LcrV+-immunized individuals (four groups of six mice) were given mAbs against CD4, CD8 or CD25
lymphocyte subsets or an isotype (control) mAb; the latter was also administered to a group of four LcrV -immunized mice. Next, all groups of mice were intravenously
inoculated with 1  103 Y. pseudotuberculosis. Bacteria in the spleen and liver of each animal were counted 5 days later. Bars indicate the median bacterial count in each organ.
⁄
, p < 0.05 vs. control-vaccinated mice in a one-way analysis of variance.

Fig. 3. Y. pseudotuberculosis growth in mice after adoptive transfer of serum from LcrV+ lactococci-immunized mice. Four naive BALB/c mice were intravenously infused with
300 lL of serum from LcrV+ (immunized) or LcrV (mock) lactococci-immunized BALB/c mice. Twenty-four hours later, recipients were intravenously inoculated with 1  103
Y. pseudotuberculosis. A. The anti-LcrV IgG titer in transferred serum. Bars represent the mean ± SD from four individuals. B. Yersinia counts in the spleen and liver of each
recipient mouse at day 5 post-challenge. Bars represent the median values; bacteria in organs were not counted in an LcrV lactococci-immunized mouse that dies on day 5
(indicated by crossbones). ⁄, p < 0.05 vs. control-vaccinated mice in a one-way analysis of variance.
5766 C. Daniel et al. / Vaccine 34 (2016) 5762–5767
was not observed in the spleen. Given that there are similar num-
bers of resident macrophages in the spleen and the liver [21,22],
better hepatic clearance of opsonized Yersinia is probably related
to greater activity by hepatic phagocytes. This difference might
be related to the expression level of the membrane Fcc receptor,
which is known to be upregulated during infection [23]. Further-
more, elimination of bacteria by Kupffer cells depends on the inter-
action with neutrophils, which migrate rapidly to the liver in
response to an infection [24]. Alternatively, polymorphonuclear
recruitment might be more intense in the liver than in the spleen.
Taken as a whole, these data suggest that the cellular response has
a greater role than the humoral response in Yersinia clearance in
mice immunized with Ll-LcrV.
Lastly, we tested the ability of the Ll-LcrV strain to sustain long-
term protection against Y. pseudotuberculosis. In groups of ten
immunized BALB/c mice, we assessed survival in response to a nor-
mally 100% lethal Y. pseudotuberculosis intravenous challenge
(1  103 bacteria) 6–9 months after the first vaccine administra-
tion. Serum anti-LcrV IgG was quantified as a marker of immuniza-
tion. As shown in Fig. 4, antibody levels were maintained over
time. However, the protective immunity conferred by LL-LcrV
decreased slightly over time; nevertheless (and in the absence of
a booster dose), almost 60% of the mice survived a lethal bacterial
challenge at 9 months post-vaccination. These findings suggest
that LcrV-specific antibodies in the serum may not be the most
important factors in conferring long-term, protective immunity.
In our previous work on the L. lactis Ll-LcrV strain, we had
shown that high titers of LcrV-specific systemic IgG and mucosal
IgA antibodies were detected at day 53 after immunization. More-
over, the amounts of IL-2, IL-10 and IFN-c secreted by cultured
splenocytes after stimulation by LcrV were found to be signifi-
cantly higher for Ll-LcrV than for Ll [18]. In the present study, we
extended our observations and demonstrated that both antibody-
and cell-mediated immune responses are needed for LcrV-
specific protection of mice against a systemic challenge with Y.
pseudotuberculosis. This is the first study to have characterized
the LcrV-induced immune protection against Y. pseudotuberculosis.
A few recent studies have shown that NK and CD8+ T cells help to
protect against systemic and oral Y. pseudotuberculosis infections
[25–27]. In this context, a YopE epitope was found to a major
CD8+ T cell antigen during the primary intragastric infection of
mice with Y. pseudotuberculosis [26,28]. In view of these data,
Fig. 4. Persistence of specific immunity after vaccination with LcrV+ L. lactis. Groups of 10–11 BALB/c mice were intranasally immunized with LcrV+ or LcrV (mock) L. lactis
and then intravenously inoculated with 1  103 Y. pseudotuberculosis at day 53, 3 months or 9 months post-immunization. All animals were monitored daily for 3 weeks.
Survival and the associated anti-LcrV IgG blood titer (measured at the time of challenge) are shown. Bars represent the mean ± SD antibody titers.
expression of this YopE epitope in Ll-LcrV might stimulate CD8
responses and improve the protective immunity conferred by
intranasal Lactococcus vaccination.
A number of vaccines that variously use DNA, microparticles,
Gram-positive enhancer matrix and immunoadjuvants to deliver
LcrV from Y. pestis (a clone that has recently emerged from Y. pseu-
dotuberculosis) are known to elicit CD8+ and/or CD4+ T cells pro-
ducing IFN-against specific epitopes of LcrV; it is clear that CD8+T
and/or CD4+ IFN-c+ cell immune responses are involved in LcrV-
specific, vaccine-elicited protection [29–33]. Interestingly, we
found similarities between the immune responses triggered by
our vaccine and by Y. pestis LcrV-mediated vaccines. Immunization
of mice with Ll-LcrV elicited CD4+ T lymphocytes and CD8+ T cells
(Fig. 1). Furthermore, LcrV vaccine-induced protection was con-
ferred by CD4+ T lymphocytes and CD25+ T cells (activated CD4+
T cells and/or regulatory T cells) but not by CD8+ T cells (Fig. 2).
However, the differences observed between our LcrV vaccine and
Y. pestis-based vaccines might be due to differences in T cell
epitopes [34–37], immunization routes and delivery vectors
[29–33,38].
Many researchers have shown that antigen-producing L. lactis
can be successfully used as a mucosal vaccine vector to protect
against pathogens [17]. However, very few studies have deter-
mined which immune cells are specifically elicited and confer pro-
tection against pathogens [39–42]. Overall, the results of studies
using L. lactis as a vehicle for various antigens (Leishmania antigen
LACK, HIV-1 Gag-p-24 antigen, listeriolysin O Listeria monocytoge-
nes antigen and Giardia lamblia cyst wall protein) and the present
study demonstrate that L. lactis is able to induce a broad, protective
immune response after immunization via different administration
routes (intranasal, oral, intraperitoneal or subcutaneous). Further-
more, we and others have shown that CD8+ and/or CD4+ T cell
immune responses are clearly involved in the protection of mice
immunized with L. lactis vaccines.
Taken as a whole, these experiments demonstrated that vacci-
nation of mice with Ll-LcrV induces cell- and antibody-mediated
protective immunity against Y. pseudotuberculosis infection in the
mouse and the protection is long-lasting. We are now seeking to
determine whether our vaccination protocol primes memory T
cells to rapidly produce IFN-c and/or TNF in response to an infec-
tious challenge, thereby promoting the bactericidal capacities of
phagocytes and/or protecting these cells from the harmful effects
 C. Daniel et al. / Vaccine 34 (2016) 5762–5767
of Y. pseudotuberculosis. Having demonstrated that protection
involves CD4 T cells, we now consider that the associated LcrV-
specific T cell epitopes must be identified.
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