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Infection and Immunity, October 1999, p. 5500-5507, Vol. 67, No. 10
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rational Live Oral Carrier Vaccine Design by
Mutating Virulence-Associated Genes of Yersinia
enterocolitica
Emeka I.
Igwe,
Holger
Rüssmann,
Andreas
Roggenkamp,
Annette
Noll,
Ingo B.
Autenrieth, and
J.
Heesemann*
Max von Pettenkofer Institute for Hygiene and
Medical Microbiology, Ludwig Maximilians University Munich, 80336 Munich, Germany
Received 10 May 1999/Returned for modification 5 July 1999/Accepted 21 July 1999
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ABSTRACT |
Three different Yersinia enterocolitica serotype O8
strains harboring mutations in virulence-associated genes coding for
Yersinia adhesin A (YadA), Mn-cofactored superoxide
dismutase (SodA), and high-molecular-weight protein 1 were analyzed for
their ability to colonize and persist in tissues after orogastric
immunization of C57BL/6 mice. We demonstrated that all three
Yersinia mutant strains were markedly impaired in their
ability to disseminate into the spleens and livers of immunized mice
but were able to colonize the Peyer's patches for at least 12 days,
resulting in the induction of significant antibody titers against
Yersinia outer proteins (Yops) and in the priming of
Yersinia antigen-specific CD4+ Th1 cells
isolated from spleens. The high level of attenuation did not diminish
the immunogenic properties of the mutant strains. In fact, mice
immunized with a single oral dose of any of the mutant strains were
protected against a lethal oral-challenge infection with wild-type
Y. enterocolitica. Moreover, adoptive transfer of
Yersinia-specific antibodies from sera of mice immunized with the mutant WAP-314 sodA revealed that this protection
could be mediated by Yersinia-specific immunoglobulins.
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TEXT |
Live replicating bacteria are being
considered as attractive antigen delivery vectors. A variety of
attenuated Salmonella typhimurium, Yersinia
enterocolitica, Mycobacterium tuberculosis, and
Listeria monocytogenes mutant strains have been evaluated as
potential carrier vaccines to present heterologous antigens to the
immune systems of vaccinated mice (1, 12, 14, 25, 33).
Despite the progress in the development of new bacterial live carrier
vaccines, it has become increasingly clear that new strategies are
needed. For example, instead of knocking out genes that result in
auxotrophic mutations (e.g.,
aroA or
aroCD)
(9, 23, 48) or interference in global gene expression and
regulation (e.g.,
phoP or
phoQ) (16,
22), an attractive alternative might be to mutate genes that code
for virulence-associated factors of bacteria, leading to newly designed
vector strains with tissue tropism and restriction.
Y. enterocolitica causes enteritis and lymphadenitis in
humans and rodents (17). In mice, yersiniae preferentially
bind to M cells, thereby promoting bacterial uptake and transepithelial transport to the Peyer's patches. Both dissemination into the spleen
and liver and further proliferation within these organs mark the
initiation of a symptomatic infection. The virulence is controlled by
chromosomally encoded (Inv, Ail, and the siderophore yersiniabactin)
and plasmid-encoded (Yersinia outer proteins and Yersinia adhesin A) determinants (11). These
virulence factors and the pathogenesis of Y. enterocolitica
have been extensively studied (5, 19, 24, 38-40).
Y. enterocolitica has evolved a strategy to survive and
multiply within the lymphoid tissue, predominantly extracellularly (27, 29, 44). This strategy might be an advantageous feature for a carrier vaccine strain. The extracellular location may help the
host's immune system to eliminate the recombinant strain after a
decent time interval post-oral immunization and thus prevent a chronic colonization.
In our laboratory, we have previously described three Y. enterocolitica O8 mutant strains (34, 35, 37): (i) the
yadA-2 mutant, obtained by substituting tyrosine residues
for two histidine residues in the YadA protein, which is a
plasmid-encoded surface protein that mediates binding to
extracellular-matrix proteins, adherence to host cells, and resistance
to complement lysis and is essential for virulence of yersiniae; (ii)
the Mn-cofactored superoxide dismutase (sodA) mutant, which
is deficient in resistance to exogenous oxygen radicals produced by
phagocytes; and (iii) the irp-1 mutant, lacking the
384.6-kDa high-molecular-weight protein 1, which is part of the
siderophore yersiniabactin biosynthesis apparatus. The aim of this
study was to assess the capacity of these three isogenic Y. enterocolitica O8 strains carrying mutations in
virulence-associated genes to act as potential live oral vaccine candidates in mice.
The Yersinia strains used in this study and their
construction were described previously (34, 35, 37). Strain
WA-314 is a clinical isolate of Y. enterocolitica serotype
O8 and bears the virulence plasmid pYVO8. This isolate was used as the
parental strain for the construction of WA(pYVO8-A-2) and WA-314
sodA. Strain WA(pYVO8-A-2) was constructed by site-directed
mutagenesis, resulting in the substitution of tyrosine residues for
histidine-156 and histidine-159 of the YadA protein. In strain WA-314
sodA, the wild-type sodA gene has been replaced
by sodA::Km. To construct WA irp1, the
previously described cointegrant pRK290B8-5::pO8 was
mobilized into the virulence plasmidless mutant WA-CS
irp1::Kanr (20).
The significance of the differences among control and experimental
groups in all experiments was determined by the Student t
test. P values of <0.05 were considered statistically significant.
Determination of the course of colonization and persistence in
mouse tissues.
The virulence of the Y. enterocolitica
mutant strains was tested in the orogastric mouse infection model as
described previously (37). Prior to infection of 6- to
8-week-old C57BL/6 mice, Yersinia stock suspensions were
thawed and washed twice in sterile phosphate-buffered saline (PBS; pH
7.4). After appropriate dilution, bacteria were fed to groups of eight
C57BL/6 mice by the use of a microliter pipette. The actual number of
bacteria administered was determined by plating serial dilutions on
Mueller-Hinton agar and counting CFU after incubation for 36 h at
27°C. Control mice were given an equal volume of sterile PBS. At
various days postinfection (p.i.), mice were sacrificed. After aseptic
removal of the organs, the Peyer's patches, spleen, and liver of each
mouse were homogenized in 1, 5, and 5 ml, respectively, of sterile PBS
containing 0.1% Tergitol TMN 10 (Fluka, Buchs, Switzerland) and 0.1%
bovine serum albumin (E. Merck AG, Darmstadt, Germany) by the use of
tissue homogenizers, whereas the small intestine was washed with 10 ml of ice-cold PBS.
The course of immunization was determined by counting the numbers of
surviving bacteria, as CFU, in the lumen of the small intestine, the
Peyer's patches, the spleen, and the liver on days 2, 5, 7, 12, and 21 postimmunization. The results are summarized in Fig.
1. Two days after orogastric
immunization, the mutant strains and the wild-type strain colonized the
small intestine and the Peyer's patches (Fig. 1A). The course of
infection with WA-314 was progressive, with dissemination of the
bacteria into the spleen (mean ± standard deviation, 5.7 × 105 ± 5.5 × 105 CFU) and the liver
(5.0 × 105 ± 5.1 × 105 CFU)
by day 5 (Fig. 1B). At this time point, only the mutant strain
WA(pYVO8-A-2) was detected in the spleen (2.2 × 102 ± 0.9 × 102 CFU) and the liver
(2.4 × 102 ± 2.5 × 102 CFU),
whereas no dissemination of WA-314 sodA and WA
irp1 into these organs was observed. In contrast, all three
mutant strains profoundly colonized the gut and the Peyer's patches
(Fig. 1B). On day 7 p.i., half of the mice infected with the
wild-type Y. enterocolitica strain died due to the high
bacterial load in the spleen and liver leading to a septic course of
infection (Fig. 1C). In contrast, bacterial counts of WA(pYVO8-A-2) in
the spleen (3.1 × 103 ± 3.3 × 103 CFU) and liver (1.8 × 103 ± 2.1 × 103 CFU) were more than 100 times lower than
those of the wild-type strain. The mutant strain WA-314 sodA
colonized both organs in smaller numbers (10 to 200 CFU per organ),
whereas WA irp1 could not be reisolated from the spleen or
liver throughout the investigated period of time, although this strain
was able to colonize the Peyer's patches (1.7 × 104 ± 0.5 × 104 CFU on day 7).
While all mice infected with wild-type strain WA-314 died between days
6 and 10 p.i., all mice immunized with mutant strains showed
markedly reduced signs of illness and survived. By day 12 p.i.,
WA(pYVO8-A-2) and WA-314 sodA were eliminated from the
spleens and livers of immunized mice (Fig. 1D). The latter strain was
reisolated from Peyer's patches (3.5 × 103 ± 3.5 × 103 CFU) and the small intestine (3.4 × 103 ± 3.6 × 103 CFU) in numbers
that were 10 times higher than those for WA(pYVO8-A-2) or WA
irp1. Twenty-one days after oral inoculation of the mutant strains, only WA-314 sodA was still able to colonize the
Peyer's patches, although it did so in small numbers (~40 CFU per
organ). In addition, this mutant strain was reisolated from the small intestine at a 100-fold-higher concentration (3.2 × 103 ± 3.3 × 103 CFU) than
WA(pYVO8-A-2) or WA irp1. Thus, all three mutant strains were able to colonize the Peyer's patches of immunized C57BL/6 mice,
to different degrees, for at least 2 weeks but were markedly impaired
in their ability to disseminate into the spleen and liver compared to
the fully virulent parental strain.

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FIG. 1.
Time course of colonization and persistence of Y. enterocolitica in the liver (L), spleen (SP), Peyer's patches
(PP), and small intestine (SI). C57BL/6 mice were orally immunized with
108 Y. enterocolitica O8 mutant or isogenic
wild-type organisms. Two, 5, 7, 12, and 21 days later, the mice were
killed and the numbers of bacteria (CFU) present in the different mouse
tissues were determined. Four of the eight mice immunized with
Yersinia wild-type strain WA-314 succumbed on day 7 p.i., whereas the rest of the group died between days 8 and 10. Values
are means for eight animals, with standard errors of the means
indicated by error bars. *, value differs from that of mice infected
with the Yersinia wild-type strain (P < 0.05).
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Antibody responses against Yersinia outer
proteins.
In the next set of experiments, it was investigated
whether the Yersinia mutant strains were able to elicit
humoral immune responses against Yersinia outer proteins,
for which the acronym Yop is used. Yersinia-specific
anti-Yop antibodies in sera of immunized mice were detected by a
Yersinia-specific enzyme-linked immunosorbent assay (ELISA)
as described previously (21, 28, 42). Yersinia
outer proteins, at a concentration of 10 µg/ml in PBS, were used to
coat 96-well microtiter plates (Greiner, Frickenhausen, Germany).
Serial dilutions of sera from each of eight mice per group were carried
out in PBS containing 0.5% Tween 20 (Merck, Darmstadt, Germany) and
2% bovine serum albumin BSA. Alkaline phosphatase-conjugated
anti-mouse immunoglobulin G (IgG), IgA, and IgM (Sigma, Deisenhofen,
Germany) were diluted 1:1,000 with PBS containing 0.5% Tween 20 and
used as secondary antibodies. Disodium
p-nitrophenylphosphate (Sigma) was used as the substrate. Optical densities were measured with an ELISA reader (Flow
Laboratories, Meckenheim, Germany) at a wavelength of 405 nm. Five
duplicates of sera from nonimmunized control mice were tested as
negative controls to obtain cutoff values. The cutoff value in this
study was defined as the mean absorbance of the negative controls plus 2 standard deviations.
Groups of five mice were immunized with a single oral dose of
10
8 organisms of one of the various strains, and blood
samples were
collected on days 7, 12, 23, 35, and 90 after
immunization. The
results are shown in Fig.
2. All mice immunized with the mutant
strains showed the highest serum IgA and IgM antibody titers on
day
12 p.i. and the highest serum IgG antibody titers on day 23
p.i. Thereafter, a continuous decline of the titers was observed.
Over
the course of 90 days, the mutant strains differed in the
magnitude of
Yersinia-specific IgG, IgA, and IgM responses elicited
in
sera of mice. On day 23, WA(pYVO8-A-2) elicited a 23-fold-higher
titer
(1:14,000) and WA-314
sodA elicited a 17-fold-higher titer
(1:10,000) of
Yersinia-specific serum IgG antibody than WA
irp1 (1:600) (
P < 0.05) (Fig.
2). Mice
immunized with WA-314
sodA elicited
a 10-fold-higher titer
(1:600) of serum IgG antibody than those
given WA(pYVO8-A-2) (1:60) 90 days after the immunization (
P <
0.05). At this time
point, significant
Yersinia-specific IgG titers
were no
longer detectable in sera from mice immunized with WA
irp1.

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FIG. 2.
Serum IgG, IgA, and IgM antibody responses of C57BL/6
mice prior to immunization (day 0) and 7, 12, 23, 35, and 90 days after
oral immunization with 108 organisms of the indicated
Yersinia mutant strains, as determined by using a
Yersinia outer protein-specific ELISA. Columns represent
means and standard deviations of results (log10 titer)
obtained from eight mice. *, value differs from that of a control
serum obtained prior to oral immunization (P < 0.05).
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Induction of Yersinia-specific splenic T cells.
To
investigate the abilities of the three mutant strains to elicit
Yersinia-specific T-cell responses, mice were orally
immunized with 108 yersiniae. Eight days after
immunization, spleens were removed and single-cell suspensions were
prepared as described previously (4). Purified T cells
(26) were stained with a fluorescein isothiocyanate-coupled
anti-CD3
(145 2C11) (Becton Dickinson, Heidelberg, Germany)
monoclonal antibody (MAb) and analyzed by flow cytometry (Epics
XL-MCL; Coulter Electronics, Krefeld, Germany) as described previously
(31). For proliferation assays, 2 × 105
purified T cells, 2 × 105 irradiated (3,000 rads)
syngeneic splenic cells, as antigen-presenting cells, and 10 µg of
antigen/ml were incubated in 96-well microtiter plates (Nunc,
Wiesbaden, Germany) with 200 µl of culture medium per well
(Click-RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2 mM
L-glutamine, HEPES, 5 × 10
5 M
2-mercaptoethanol, 10 µg of streptomycin/ml 100 U of penicillin/ml and 10% heat-inactivated fetal calf serum). The following antigens were used at a concentration of 10 µg/ml of culture medium:
heat-killed whole bacterial cells of Y. enterocolitica O8
(4), recombinant purified Yersinia heat shock
protein 60 (HSP60) (31), and recombinant purified
Yersinia outer proteins (YopD, -E, -H, and V antigen) (36). After incubation for 3 days, the cultures were pulsed with [3H]thymidine and the uptake of
[3H]thymidine was determined with a liquid scintillation
counter (Pharmacia) (31). Proliferative responses were
expressed as stimulation indices (SI), which were calculated as
follows: SI = [3H]thymidine uptake (in counts per
minute) in the presence of the indicated
antigen/[3H]thymidine uptake in the absence of that
antigen. All experiments were repeated at least three times for verification.
The proliferation of T cells depended on the presence of
antigen-presenting cells (APC) and antigen. Heat-killed
Y. enterocolitica,
Yersinia heat shock protein (HSP60),
and recombinant
Yersinia outer proteins (YopD, YopE, YopH,
and V antigen) induced significant
proliferative responses, as
summarized in Fig.
3. Upon antigenic
stimulation, T cells isolated from mice immunized with WA(pYVO8-A-2)
or
WA-314
sodA showed proliferative responses almost equivalent
to those of T cells from mice infected with
Yersinia
wild-type
strain WA-314. In contrast, T cells obtained from mice
immunized
with WA
irp1 exhibited significantly weaker
proliferative responses
to the different antigens (
P < 0.05) than T cells isolated from
mice inoculated with either of
the two other
Yersinia mutant strains
investigated in this
study. Comparisons of proliferative responses
to different
Yersinia antigens revealed distinct patterns. T cells
showed
only a weak proliferative response to HSP60, YopE, and
the V antigen
(SI < 10), whereas stimulation with YopH induced
a moderate
proliferation (SI = 10 to 20). A strong T-cell proliferative
response was observed upon stimulation with heat-killed
Y. enterocolitica or YopD (SI = 25 to 45).

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FIG. 3.
Proliferative T-cell responses to various
Yersinia-specific antigens after oral immunization with
wild-type or mutant Yersinia strains. Splenic T cells were
stimulated with 10 µg of heat-killed Y. enterocolitica
(HKY), Yersinia heat shock protein (YHSP60), and recombinant
Yersinia outer proteins (YopD, YopE, YopH, and the V
antigen) or without Yersinia antigen. Proliferative
responses are expressed as SI. Solid bars represent SI values of T
cells stimulated with the indicated antigen, whereas open bars
represent SI values of T cells exhibiting non-antigen-stimulated
(spontaneous) proliferation. Values are the means of triplicate
cultures, with standard deviations indicated by error bars. *, value
differs from that of the control (P < 0.05).
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Cytokine production by Yersinia-specific T cells.
For determination of cytokine production, the supernatants of T cells
stimulated with heat-killed Yersinia organisms were collected and used in cytokine assays. Gamma interferon (IFN-
) levels were determined by capture ELISA as described previously (2, 4). Briefly, ELISA microtiter plates were coated with anti-IFN-
MAb (AN-18.17.24). Biotin-labeled anti-IFN-
MAb
(R4-6A2) and avidin-biotin-alkaline phosphatase complex (Vectastain
ABC-AP kit; Camon Wiesbaden, Germany) were used, and the optical
densities at wavelengths of 405 and 490 nm were determined with an
ELISA reader. In parallel, an interleukin-4 (IL-4)-specific ELISA,
including the anti-IL4 MAbs 11B11 (biotin labeled) and BVD6 24G2, was
carried out as described for IFN-
.
Determination of IFN-

levels revealed that restimulated T cells from
mice immunized with the mutant strains produced significant
quantities
of IFN-

by day 8 after oral immunization compared
to the control
group of nonrestimulated T cells from immunized
mice (
P < 0.05) (Fig.
4). However, the amounts
of IFN-

produced
by T cells from mice immunized with the mutant
strains were lower
than those of mice immunized with the wild-type
Y. enterocolitica strain. In contrast, no significant
quantities of IL-4 were detected
after immunization with the wild-type
or mutant strains (Fig.
4).

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FIG. 4.
IFN- and IL-4 production by splenic T cells of mice
after oral immunization with wild-type or mutant Y. enterocolitica strains. T cells were stimulated with 10 µg of
heat-killed Y. enterocolitica (HKY) per ml. Supernatants
were used in an IFN- - and IL-4-specific ELISA. The optical density
values revealed in the ELISA are expressed as units of IFN- and IL-4
per milliliter according to the linear portion of the standard curve.
Results are the means ± standard deviations (error bars) of
values for five animals. *, value differs from that of the control
(nonstimulated T cells) (P < 0.05).
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Protective immunity.
Groups of eight mice were orally
immunized with a single dose of 108 attenuated
Yersinia mutant organisms. Ten weeks after this
immunization, mice were challenged with 5 × 108
wild-type Y. enterocolitica WA-314 organisms (10 times the
50% lethal dose [LD50]) by the oral route. To determine
the extent of protection, the bacterial loads in the lumen of the small
intestines, the Peyer's patches, the spleens, and the livers of
infected animals were determined as described above.
In comparison to the control group of nonimmunized mice, no signs of
illness were observed in mice immunized with the attenuated
Yersinia strains. Five days after the oral challenge, mice
were
sacrificed and the colonization and persistence of wild-type
Y. enterocolitica WA-314 in vivo was investigated (Table
1). Three
of the eight mice in the
nonimmunized group died due to the challenge
on day 5. In the remaining
five mice, wild-type yersiniae were
present in large numbers in the
lumen of the small intestine (3.4
× 10
5 ± 1.7 × 10
5 CFU), the Peyer's patches (1.8 × 10
8 ± 0.9 × 10
6 CFU), the spleen
(4.1 × 10
5 ± 0.8 × 10
5 CFU),
and the liver (5.9 × 10
4 ± 1.2 × 10
4 CFU). In contrast, the wild-type
Y. enterocolitica strain was
not isolated from the spleen or the
liver of any immunized mouse.
In addition, mice immunized with
WA(pYVO8-A-2) or WA-314
sodA had 1,000- to 10,000-fold-lower
bacterial counts in the small
intestine and the Peyer's patches than
nonimmunized mice (
P <
0.05). In mice immunized with
WA
irp1, 100- to 1,000-fold-lower
numbers of wild-type
Yersinia were detected in the latter organs
(
P < 0.05).
Adoptive transfer of Yersinia-specific antibodies.
To determine whether Yersinia antiserum from mice orally
immunized with the Yersinia WA-314 sodA mutant
strain can mediate protection against an intravenous challenge with a
lethal dose of wild-type Y. enterocolitica,
adoptive-transfer experiments were carried out. Four weeks after oral
inoculation of 108 WA-314 sodA organisms,
hyperimmune sera from mice were collected. After ammonium sulfate
precipitation, the immunoglobulins were dialyzed against PBS overnight
at 4°C and the protein content was determined as described previously
(49). C57BL/6 mice (eight per group) were each treated
intravenously with 400 µg of a mouse immunoglobulin-rich fraction 1 day prior to an intravenous (i.v.) challenge with 10 times the
LD50 of Y. enterocolitica O8 wild-type strain
WA-314. Five and 11 days after the challenge, bacterial counts in
spleens of mice were determined.
As demonstrated in Table
2, the group of
mice treated with the WA-314
sodA antiserum showed a
100-fold-lower bacterial load
in their spleens than the group of mice
treated with the purified
serum from naive mice (
P < 0.05) 5 days after the challenge. Moreover,
in the former group,
all mice survived the
Yersinia infection
and no bacteria
were isolated from spleens on day 11 after the
challenge, whereas all
mice of the latter group died between days
6 and 9 p.i.
Y. enterocolitica has been recognized as a potential
bacterial live carrier by several groups. Sory et al. made use of
Y. enterocolitica strains to induce mucosal and serum
antibody responses
against the cholera toxin B subunit and the
cytoplasmic CRA protein
of
Trypanosoma cruzi (
46,
47). However, in those studies,
the authors did not carry out the
experiments with attenuated
Yersinia strains but rather used
Y. enterocolitica serotype O9
strains (biotype II), which
are less virulent in mice (low-pathogenicity
group) than those of
serotype O8 (biotype I B; high-pathogenicity
group). In contrast, Bowe
et al. (
7) and O'Gaora et al. (
32,
33)
constructed a highly attenuated
Y. enterocolitica O8
aroA mutant strain which was found to persist in the
Peyer's patches,
mesenteric lymph nodes, spleen, and liver for only 3 days after
oral infection. Consequently, mice immunized orally with a
single
dose of this mutant strain were not protected against a lethal
wild-type infection. More recently, Dorrell et al. described a
Y. enterocolitica O8
ompR mutant strain which did not
cause a
lethal course of infection in orally immunized BALB/c mice
(
13).
Spleens and livers of infected mice were colonized
with the mutant
strain for 21 days. Mice orally immunized with a single
dose of
the O8
ompR mutant strain were partially protected
against an
oral challenge with the virulent
Y. enterocolitica parent
strain.
The
Yersinia mutant strains investigated in this study were
capable of translocating from the intestinal lumen to the Peyer's
patches, where they persisted for at least 12 days. All three
mutant
strains were markedly impaired in their ability to disseminate
from
Peyer's patch tissue into the spleen and liver, resulting
in survival
of all infected mice. The
yadA-2 mutant strain WA(pYVO8-A-2)
was reisolated from these organs on days 5 and 7 p.i. 100 times
less than the isogenic wild-type strain, whereas the mutant strain
WA-314
sodA was detected in the spleen and liver only on day
7
p.i. even 1,000 times less than the wild-type
Yersinia strain.
Surprisingly, the mutant strain WA
irp1 did not colonize the latter
organs at any time during
the course of
immunization.
A
yadA null mutant of
Y. enterocolitica is
characterized by an impaired ability to colonize the intestinal mucosa
(
27).
Previously, we showed that the substitution of
tyrosine residues
for two histidine residues in YadA resulted in
abrogation of binding
to various extracellular-matrix proteins
(
42,
43) and of adherence
to HEp-2 cells, whereas
autoagglutination (
45) and serum complement
resistance
(
10) remained unaffected. However, the collagen-binding
function appeared to not be required for translocation of WA(pYVO8-A-2)
from the gut lumen to the Peyer's
patches.
The attenuation of
Y. enterocolitica resulting from the
mutation of the
sodA gene is due to the additive effects of
the reduced
detoxification abilities of metabolically produced
bacterial superoxide
and of the increased susceptibility to killing by
polymorphonuclear
leukocytes (
35). The
sodA gene
was found to be upregulated under
conditions of aerobiosis and iron
starvation in
Escherichia coli (
15). Such
conditions are encountered by extracellular yersiniae
in the spleen and
liver. In contrast,
sodA is known to be downregulated
under
anaerobic or microaerophilic conditions, such as in the
gut or in
abscesses of Peyer's patches during
Yersinia infection
(
3,
17,
30). In fact, WA-314
sodA colonized the
small intestine
and the Peyer's patches for at least 3 weeks after the
orogastric
immunization, indicating that the
sodA gene is
not required for
intraluminal
growth.
Highly pathogenic
Y. enterocolitica strains possess a
chromosomal cluster of iron-regulated genes located in the
high-pathogenicity
island. This gene cluster carries genes for the
biosynthesis and
uptake of the
Yersinia siderophore
yersiniabactin (
irp1-9 and
fyuA), which is a
high-affinity ferric iron uptake system that
significantly contributes
to the virulence of yersiniae (
8,
18). WA
irp1
was able to translocate from the intestinal lumen
to the Peyer's
patches, but its abilities to cause a systemic
infection and to
colonize the spleen and liver were totally impaired.
As mentioned
above, the evasion strategy of wild-type
Y. enterocolitica in the host eventually results in extracellular survival and
multiplication
in the spleen and liver. Evidently, the siderophore
yersiniabactin
is essential for the ability of yersiniae to survive and
multiply
within these organs, whereas presumed accessory iron uptake
systems
in these organisms are sufficient to mediate survival during
the
first stage of colonization of the
host.
The investigation of humoral and cellular immune responses to
Yersinia-specific antigens elicited by the
Yersinia mutant strains
revealed that WA(pYVO8-A-2) and
WA-314
sodA were able to induce
significantly higher IgG,
IgA, and IgM antibody titers against
Yersinia outer proteins
than WA
irp1. It is conceivable that the
prolonged
colonization of the spleen and liver by the former strains
provoked a
stronger humoral immune response. On the other hand,
it appears that
the higher bacterial load of WA-314
sodA in the
Peyer's
patches and the small intestine at 21 days p.i. contributed
to the
10-fold-higher IgG antibody titer compared to WA(pYVO8-A-2)
at 90 days
p.i.
The mutant strains WA(pYVO8-A-2) and WA-314
sodA elicited
stronger cellular immune responses against a variety of
Yersinia-specific
antigens than WA
irp1. These
data suggest that the transient and
weak colonization of the spleens
and livers of infected mice by
attenuated
Yersinia strains
enhances
Yersinia-specific antibody
and T-cell responses.
However, this is not an essential prerequisite
of an effective
Yersinia live oral carrier vaccine, because WA
irp1 was also able to induce significant humoral and
cellular
immune responses against
Yersinia-specific
antigens. On the other
hand, we cannot exclude the possibility that
much larger numbers
of yersiniae eventually reached the spleen and
liver but were
rapidly killed and thus did not appear in the CFU
counting
assay.
It has been previously shown that T cells from C57BL/6 mice immunized
with a sublethal dose of wild-type
Yersinia produced
significant levels of IFN-

upon exposure to antigen (heat-killed
Y. enterocolitica), while they did not produce IL-4
(
2). T
cells from mice immunized with the
Yersinia mutant strains showed
the same pattern of cytokine
production. Thus, like wild-type
Y. enterocolitica, the
mutant strains induced pronounced Th1 responses.
IFN-

-producing Th1
cells are known to provide help for cell-mediated
immune responses
which are crucial for the defense from intracellular
pathogens
(
6).
A single oral immunization of a mouse with any of the mutant strains
resulted in full protection against a lethal wild-type
Yersinia infection. Moreover, in experiments involving
adoptive
transfer of
Yersinia-specific antibodies from sera
of mice immunized
with WA-314
sodA, we were able to
demonstrate that this protection
could be mediated by
Yersinia-specific immunoglobulins. Thus,
the high level of
attenuation did not diminish the immunogenic
properties of the mutant
strains.
The mutant
Yersinia strains investigated in this study
elicited pronounced humoral and cellular immune responses against
Yersinia outer proteins, which are effector proteins of
Yersinia's type
III secretion system. Recently,
Rüssmann et al. showed that the
delivery of viral epitopes
through the
S. typhimurium type III
secretion system
resulted in efficient stimulation of MHC class
I-restricted protective
antiviral immune responses in vaccinated
mice (
41). The use
of
Yersinia outer proteins as carriers for
heterologous
antigens in attenuated
Yersinia strains may be an
attractive
strategy to stimulate both humoral and cellular immune
responses.
We have shown, based on designed mutations of virulence-associated
genes, that rationally attenuated
Y. enterocolitica O8
strains have a great potential to serve as safe and effective
live oral
carrier vaccines for the delivery of heterologous antigens
in future
studies.
 |
ACKNOWLEDGMENTS |
E.I.I. and H.R. contributed equally to this work.
We thank Jeannette Sauer for expert technical assistance.
This work was supported by the Bayerische Forschungsförderung
(FORGEN). H.R. was supported by the AIDS-Stipendienprogramm of the
Bundesministerium für Forschung und Technologie/Germany.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Max von
Pettenkofer Institute for Hygiene and Medical Microbiology, Ludwig
Maximilians University Munich, Pettenkoferstr. 9a, 80336 Munich,
Germany. Phone: 49-89-51605200. Fax: 49-89-51605202. E-mail:
ruessmann{at}m3401.mpk.med.uni-muenchen.de.
Editor:
S. H. E. Kaufmann
 |
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Infection and Immunity, October 1999, p. 5500-5507, Vol. 67, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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