FEMS Immunology and Medical Microbiology 37 (2003) 135^145

www.fems-microbiology.org

E¡ect of in situ expression of human interleukin-6 on antibody

responses against Salmonella typhimurium antigens
Yuanyi Li, Kerstin Reichenstein, Ramona Ullrich, Tobias Danner,
Bernd-Ulrich von Specht, Heinz P. Hahn

Chirurgische Universita«tsklinik, Chirurgische Forschung, Freiburg i. Br., Germany

Received 11 September 2002; received in revised form 22 November 2002 ; accepted 10 December 2002

First published online 7 March 2003

Abstract

In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized
human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for
secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyAs ). Through stable introduction of a second hIL-6-HlyAs
expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyAs secretion efficiencies were increased by at
least 10-fold. As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did
plasmids interfere in their stabilities. Increased hIL-6-HlyAs expression did not adversely interfere with bacterial growth. Comparative
immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-
production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses. In mice
immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared
to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain. Higher sera antibody titers were accompanied by increased
numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer’s patches, respectively. These data suggest that systemic
antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of
recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses.
= 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Hemolysin; Immunomodulation; Interleukin-6 ; Protein secretion; Salmonella

1. Introduction pressed foreign antigens. Generally immune responses are

Over the past 20 years, live attenuated Salmonella
strains have been extensively studied as carriers for heter-
ologous vaccine antigens and e¡ector proteins with immu-
nomodulatory and immunoadjuvancy potential, including
mucosal adjuvant proteins and cytokines [1^3]. Although
those strains experience a drastic reduction in their path-
ogenicity, remaining virulence is still su⁄cient to trigger
e⁄ciently immune responses against recombinantly ex-
* Corresponding author : Tel. : +49 (761) 270 2743;
Fax : +49 (761) 270 2579.
E-mail address : hahn@ch11.ukl.uni-freiburg.de (H.P. Hahn).
0928-8244 / 03 / $22.00 = 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0928-8244(03)00066-X
predominantly directed against surface antigens of the Sal-
monella carrier cell, but, depending on the nature of the
antigen, protective immunity can also be induced against
the foreign protein delivered by the Salmonella cell.
As mucosal pathogens, Salmonella vaccine carriers have
the potential to induce both systemic and mucosal immune
responses [4]. Studies in mice have shown that a variety of
immune responses, including cytotoxic T cells, systemic
antibody responses, as well as delayed-type hypersensitiv-
ity, are induced by attenuated Salmonella typhimurium
strains expressing recombinant antigens [5]. Less fre-
quently mucosal immune responses, e.g. mucosal secretory
IgA responses, have been reported for a variety of anti-
gens [6]. The type of immunity induced depends heavily on
the nature of the antigen, but in most cases systemic re-
sponses dominate over mucosal responses.
One approach by which mucosal responses may be se-
lectively enhanced is through co-expression of certain im-

FEMSIM 1522 16-6-03

136 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
munocompetent Th2-type cytokines. The feasibility of this
approach has been previously shown for IL-5 using viral
and bacterial vaccine carrier systems, where an increased
mucosal immunoreactivity against the co-expressed vac-
cine antigen could be demonstrated [7^9]. Similar ap-
proaches with IL-4 and IL-6 in attenuated Salmonella
strains, however, did not con¢rm the postulated immuno-
modulatory e¡ect [10,11]. This may be partly due to the
fact that plasmid-encoded expression systems, which lead
to an intracellular arresting of the cytokines, were used. In
this case, the in vivo accessibility of the recombinant cy-
tokine depends solely on the unspeci¢c release of soluble
protein in the course of the desintegration of the Salmo-
nella cells. Furthermore, such cytokines tend to aggregate
and form inclusion bodies when overexpressed intracellu-
larly in bacterial cells, further reducing the amount of
bioactive cytokine [12^14]. Moreover, IL-6, like IL-5 and
IL-4, requires correct disul¢de bond formation for devel-
opment of full biological activity, which, because of the
reducing milieu, is impaired in the bacterial cytoplasm
[15^17]. Thus, e⁄cient extracellular cytokine delivery in
Salmonella vaccine strains should have a number of strik-
ing advantages which eventually contribute to an im-
proved e¡ect or protein action.
Secretory expression of foreign proteins in Escherichia
coli and Salmonella can be achieved with the plasmid-
borne K-hemolysin (Hly) secretion system [18^20]. As pre-
viously demonstrated for various antigens, translocation
of a foreign protein across the bacterial cell envelope by
the Hly transport machinery depends on the C-terminal
Hly secretion signal HlyAs genetically fused to the foreign
protein sequence. This secretion signal is not released dur-
ing the export process, resulting in secretion of a fusion
protein, which permanently carries a C-terminal extension
of about 60 amino acid residues. Because of its low im-
munogenicity this C-terminal peptide does not markedly
contribute to the overall B- and T-cell responses against
the hybrid vaccine antigens [19]. However, the C-terminal
HlyAs extension could in£uence bioactivity of secreted
e¡ector proteins, such as cytokines.
IL-6 is a multifunctional cytokine, which also plays a
crucial role in B-cell terminal di¡erentiation and develop-
ment of secretory IgA responses at mucosae [21]. To in-
vestigate the e¡ect of extracellular delivery of a Th2-type
cytokine on antibody responses, particularly along mu-
cosal surfaces, we previously fused the cDNA encoding
mature human IL-6 to the HlyAs secretion signal, propos-
ing that the additional IL-6 synthesis leads to a selective
increase in humoral IgA responses. Recombinant E. coli
and S. typhimurium strains expressing the hIL-6-hlyAs
gene as part of a reconstructed natural hly operon secrete
relevant amounts of a bioactive hIL-6-HlyAs hybrid pro-
tein into the culture supernatant [22]. More detailed struc-
ture-function analysis of the puri¢ed protein revealed that
the C-terminal fusion part apparently has no impact on
the proper protein folding and correct disul¢de bridge
FEMSIM 1522 16-6-03
formation, resulting in a secretion-competent hIL-6 fusion
protein, indistinguishable in its bioactivity from mature
recombinant hIL-6 [23]. Immunization studies in mice
with this hIL-6-expressing Salmonella strain, however, re-
vealed no signi¢cant enhancement of antibody responses
against bacterial antigens (unpublished results). Assuming
that secreted hIL-6 amounts were too low to trigger an in
vivo e¡ect, we pursued di¡erent strategies to increase hIL-
6-HlyAs secretion e⁄ciencies in the previously constructed
S. typhimurium vaccine strains [23].
Here, we report the construction of an optimized S.
typhimurium strain, with an about 10-fold improved secre-
tion e⁄ciency for hIL-6-HlyAs . Comparative immuniza-
tion studies in mice with this strain demonstrated that
the in vivo synthesis of hIL-6 induces increased systemic
IgG and IgA responses against bacterial lipopolysaccha-
ride (LPS) upon oral administration of the vaccine strains,
suggesting that IL-6 plays a crucial role in the develop-
ment of LPS-speci¢c antibody responses. Moreover, data
support that both hIL-6 amounts and cellular location of
the recombinantly expressed hIL-6 are critical for maximal
immunomodulatory action of the e¡ector protein.
2. Materials and methods
2.1. Bacterial strains, culture conditions and plasmids
Plasmids pCH2G and pCH1A and S. typhimurium
strain X4064 have been previously described [22], while
plasmids pTrc99a and pACYC184 were from Amersham
Biosciences and New England Biolabs, respectively. Plas-
mid-bearing strains of S. typhimurium and E. coli XL1-
blue were grown at 37‡C in Luria^Bertani (LB) broth,
supplemented with 100 Wg ml31 ampicillin or 50 Wg ml31
chloramphenicol. If required, isopropyl-L-D-thiogalacto-
pyranoside (IPTG) was added to E. coli cultures for
gene induction. Transformation of S. typhimurium and
E. coli was achieved by standard electroporation protocol
[24]. For co-transformation, cells were electroporated with
a mixture of both plasmids. For immunization, Salmonella
strains were grown at 37‡C in 100 ml of LB broth with
ampicillin to an OD578 of 1.2^1.5. Cells were washed once
resuspended in 3 ml of 5%(w/v) sodium bicarbonate. Bac-
terial suspensions were adjusted to 5 OD578 /ml, which cor-
responds to approximately 1U1010 cells ml31 , and kept on
ice until administration to the mice. To recover Salmonella
from spleen or liver, organ samples were homogenized in
phosphate-bu¡ered saline (PBS) using a Wheaton homo-
genator and plated onto LB or xylose-lysine-deoxycholate
agar (Merck Eurolab) with the appropriate antibiotics.
2.2. LPS and cell homogenate preparation
Cell homogenates were prepared from stationary-phase
S. typhimurium X4064 cells by standard sonication proce-
 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
dure. Homogenates were adjusted to a protein concentra-
tion of 1 mg ml31 by dilution with PBS and stored frozen
in aliquots.
LPS was isolated from freeze-dried S. typhimurium
X4064 cell pellets following the hot phenol method of
[25] with modi¢cations. Typically, 1.5 g of a freeze-dried
cell pellet was resuspended in 53 ml of water and then an
equal volume of prewarmed (65‡C) water-saturated phenol
was added. The mixture was kept at 65‡C and vigorously
mixed for 15 min. For phase separation, the mixture was
cooled down in ice and centrifuged at 3000Ug and 4‡C for
30 min. After removal of the water phase, the phenol
phase was re-extracted with the same volume of water
and the combined water extracts extensively dialyzed
against water. The freeze-dried crude LPS preparation
was redissolved in 10 ml of PBS. For further puri¢cation,
crude LPS suspension was adjusted to 10 mM MgCl2 and
incubated for 30 min at 37‡C with 10 Wg ml31 of DNase I
and RNase A each. LPS was subsequently centrifuged for
3 h at 105 000Ug and 4‡C and resuspended with vigorous
mixing in water. The LPS suspension was recentrifuged
twice and after freeze-drying suspended in PBS at a ¢nal
concentration of 10 mg ml31 . The puri¢ed LPS prepara-
tion was essentially free of any protein contaminations
and contained less than 4% of nucleic acids as judged
from silver-stained sodium dodecyl sulfate^polyacrylamide
gel electrophoresis (SDS^PAGE) analysis [26] and absorp-
tion at 260 nm, respectively. Yields of puri¢ed LPS were in
the range of 20 mg per g of freeze-dried S. typhimurium
cell pellet.
2.3. Plasmid constructions
Recombinant DNA techniques were performed using
standard procedures [24] with DNA-modifying enzymes
used in accordance with the supplier’s instructions (New
England Biolabs). To overexpress hIL-6-HlyAs fusion pro-
tein, we previously constructed plasmid pYL1E by insert-
ing a polymerase chain reaction (PCR)-generated 852-bp
fragment encoding the complete hIL-6-hlyAs sequence be-
tween the NcoI and HindIII site of pTrc99a vector (un-
published data). In pYL1E the hIL-6 fusion gene is under
the transcriptional control of the Ptrc promoter and a
downstream terminator, forming an hIL-6-hlyAs expres-
sion cassette. The cassette was released from pYL1E by
digestion with ScaI and SfoI and the resulting 2072-bp
fragment ligated into the EcoRV site of pACYC184. Plas-
mid clones with fragment oriented in the opposite tran-
scriptional orientation of the Cm resistance gene were
identi¢ed by characteristic EcoRI and NcoI restriction pat-
tern and further analyzed for IPTG-inducible expression
of the fusion gene in E. coli. The resulting plasmid con-
struct pYL3E was introduced either alone or in combina-
tion with pCH1A and pCH2G, respectively, into S. typh-
imurium X4064 or E. coli.
FEMSIM 1522 16-6-03
137
2.4. SDS^PAGE and Western blot analysis
SDS^PAGE (12% PAA gels) and Western immunoblot-
ting were performed as described earlier [3], using hIL-6-
speci¢c IgG1 -type mAb (RpD Systems) in a 1:250 dilu-
tion for immunodetection. For colorimetric detection,
goat anti-mouse IgG (whole molecule) conjugated to alka-
line phosphatase (Sigma-Aldrich) was applied in a 1:2000
dilution, followed by standard NBT/BCIP color develop-
ment. To increase the detection sensitivity, blots were sub-
sequently probed for a 2-h period with a 1:2000 dilution
of rabbit anti-mouse IgG (whole molecule)-horseradish
peroxidase conjugate followed by chemoluminescence de-
tection as described earlier [23]. For preparation of cell-
free culture supernatant, bacteria were pelleted from cul-
ture at 4300Ug for 20 min at 4‡C and supernatants con-
centrated under vacuum prior to sample denaturing and
loading to the gel.
2.5. Sera and lavage samples
Blood samples were either collected from the tail veins
or, for terminal bleeding, from the retro-orbital plexus.
For sera preparation, clotted blood samples were centri-
fuged at 18 800Ug and 4‡C for 15 min and sera super-
natants stored in aliquots at 320‡C.
Gut lavages were obtained by £ushing the small intes-
tine with 5 ml of cold PBS, containing 1 mg ml31 bovine
serum albumin (BSA), 0.1%(w/v) NaN3 , 0.05%(v/v) Tween
20, 0.1 mg ml31 soybean trypsin inhibitor (Serva), and
1 mM phenylmethyl sulfonyl£uoride (PMSF), the latter
added immediately prior to the use of the solution. To
prepare fecal extracts, washes were heavily vortexed and
centrifuged at 7650Ug and 4‡C for 10 min. The lavage
£uids were stored frozen at 320‡C. Prior to titer measure-
ment, lavages were re-centrifuged to remove any precipi-
tate.
2.6. Isolation of splenocytes and Peyer’s patches cells
For the preparation of splenocytes, about half of a
spleen was washed once with PBS and then passaged
through a 70-Wm-pore-size cell strainer (Falcon). Erythro-
cytes were lyzed by 5-min incubation in 5 ml of 145.5 mM
NH4 Cl/17 mM Tris-HCl (pH 7.2). Remaining single-cell
suspensions were washed once with PBS and splenocytes
¢nally resuspended in 5 ml of complete RPMI medium
(RPMI 1640 medium with glutamin supplemented with
10%(v/v) fetal calf serum, 50 U ml31 penicillin, 50 Wg
ml31 streptomycin, 24 WM 2-mercaptoethanol). From
each mouse between 5 and 7 Peyer’s patches were collected
from the small intestinal wall and single-cell suspensions
prepared from the complete Peyer’s patches as described
for the spleen cells. Cells were washed once and resus-
pended in complete RPMI medium. Prior to the ELISPOT
138 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
procedure, numbers of live cells were determined upon
trypan blue staining.
2.7. IgG- and IgA-speci¢c ELISA
Speci¢c antibody titers were measured in sera and lav-
age samples following the same sandwich ELISA protocol.
96-well plates (Falcon) were coated overnight at 4‡C with
S. typhimurium cell homogenate or LPS (100 Wl/well) di-
luted in PBS to a concentration of 5 Wg ml31 and 20 Wg
ml31 , respectively. For blocking, plates were incubated for
1 h at 37‡C with 200 Wl/well of 5%(w/v) BSA in PBS.
Plates were washed four times with washing bu¡er
(0.05%(w/v) BSA/0.05% Tween 20 in PBS) and, for further
use, dried and stored at 320‡C. Upon rehydration and
washing once with washing bu¡er, sera and lavage sam-
ples were serially diluted with 2%(w/v) BSA/0.05% Tween
20 in PBS (dilution bu¡er) in the wells (100 Wl/well) and
plates incubated for 2 h at 37‡C. Plates were washed four
times and, for detection of speci¢c immunoglobulin sub-
types, incubated with a 1:2500 dilution of peroxidase-con-
jugated rabbit anti-mouse IgG or anti-mouse IgA (Sigma-
Aldrich) in dilution bu¡er for 2 h at 37‡C. Antibody bind-
ing was calorimetrically detected by 30-min incubation
with 2 mg ml31 O-phenylenediamine and 0.03%(v/v)
H2 O2 in 17 mM citrate bu¡er. Reactions were stopped
with 50 Wl/well of 4.5 M H2 SO4 and plates read in a
Dynex (MRX) ELISA reader at 490 nm with reference
wavelength set to 620 nm. Samples were measured at least
in duplicate from individual dilutions.
To compensate for the variance in the plate reactivity
and quantify ELISA titers, on each plate six serial 1:2
(lavages) and 1:3 dilutions (sera) of a standard sera
pool, derived from the terminal bleeding of eight immu-
nized mice, were loaded. The results were expressed as
antibody units calculated from the linear range of the
standard curve obtained from the sera pool given an ar-
bitrary antibody titer of 100 000 IgG U ml31 and 10 000
IgA U ml31 . The values for a minimum of four sample
dilutions falling into the linear range of the standard curve
were calculated and the geometric mean taken as antibody
titer of the sample.
To measure ELISA titers to in situ synthesized hIL-6
in mouse sera plates were coated with 20 ng per well
of carrier-free recombinant hIL-6 (RpD Systems) and
probed for IgG antibodies.
2.8. ELISPOT assay
The numbers of Ig subtype-speci¢c antibody-secreting
cells (ASCs) present in spleen and Peyer’s patches of im-
munized mice were determined by ELISPOT techniques
[27], using Biodyne B membranes in 96-well £at-bottom
plates (Nunc). Plates were coated with 2.5 Wg per well of
crude LPS and, after blocking with complete RPMI me-
dium, splenocytes and Peyer’s patches cells, respectively,
FEMSIM 1522 16-6-03
were seeded in complete RPMI medium at the indicated
cell densities. Plates were incubated overnight at 37‡C, 5%
CO2 and membrane-bound antibodies detected by incuba-
tion with subtype-speci¢c anti-mouse Ig-alkaline phospha-
tase conjugates followed by standard colorimetric detec-
tion. Further details on the ELISPOT procedure and the
enumeration of the ASCs will be published elsewhere.
2.9. Immunization experiments
For the immunization experiments 8^10 week old fe-
male inbred BALB/c mice (Charles River Germany) were
randomly distributed into the di¡erent groups. Mice were
infected orally with a dose of approximately 2U109 Sal-
monella in 200 Wl with a gastric lavage needle 10 min after
administration of 100 Wl of 5%(w/v) sodium bicarbonate.
Prior to the immunization procedure, mice were kept for
6^8 h without water. Applied bacterial numbers were veri-
¢ed by viable counting. Mice were boosted on days 28 and
42 with the same bacterial dose administered in the pri-
mary immunization. During the experiments, mice were
fed commercial mouse chow and water ad libitum. Two
days before each immunization mice were anesthetized
lightly and blood samples taken from the tail vein. At
the end of the experiments, mice were anesthetized and
larger blood volumes taken from the retro-orbital plexus.
Thereafter, mice were terminated by cervical dislocation
and, after lavaging the lung, liver, spleen, and small intes-
tines removed and processed as described above. The ani-
mal experiments reported were conducted according to the
German animal protection law and approved by the ethics
committee of the Regierungspra«sidium Freiburg.
2.10. Statistics
The P values were determined using Student’s two-tailed
t-test (Excel; Microsoft Corporation). Immunoblots and
X-ray ¢lms were photographed with a digital camera.
Electronic images were mounted in Adobe Photoshop
and ¢nally integrated in Microsoft Power Point.
3. Results
3.1. Construction of S. typhimurium strains with improved
hIL-6-HlyAs secretion
From previous studies we knew that the expression of
the hIL-6-hlyAs gene as an integral part of the original hly
operon was the ‘bottleneck’ in secretory hIL-6-HlyAs pro-
duction in both E. coli and S. typhimurium [3]. Data sug-
gested that an increase in the hIL-6-hlyAs gene copy num-
ber should eventually boost secretory hIL-6 expression.
Based on that, we developed an improved Hly export
cloning system that is composed of two vectors, one in
which the hIL-6 fusion gene is combined with the genes
 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
Fig. 1. Comparative Western blot analysis of hIL-6-HlyAs secretion in
di¡erent plasmid-bearing E. coli strains. E. coli strains harboring plas-
mids pCH1A (1A), pYL3E (3E), pCH1A+pYL3E (1A+3E), and
pCH2G (2G) were grown under similar conditions. After 5, 7, 9, and
24 h cell-free supernatants were prepared from culture samples and ana-
lyzed by hIL-6-speci¢c Western immunoblotting followed by colorimet-
ric detection of antibody binding. 200 Wl of culture supernatant was
loaded in each lane. M, pre-stained standard proteins; S, mature hIl-6
(5 ng). The upper arrow marks the full-length hIL-6-HlyAs protein.
encoding the Hly transporter apparatus (pCH1A) and a
second in which hIL-6-hlyAs is under transcriptional con-
trol of the Ptrc promotor (pYL3E). To adjust the hIL-6-
HlyAs expression to a medium level and thus minimize the
risk of intracellular precipitation of the hIL-6 fusion pro-
tein and avoid plasmid stability problems we used a rather
low copy-number vector as carrier for the additional hIL-
6-HlyAs gene copy. Plasmids pCH1A and pYL3E as well
as the corresponding control plasmids were simultaneously
introduced into E. coli and S. typhimurium by electropo-
ration.
To evaluate the e¡ect of the additional hIL-6-hlyAs gene
copy on the fusion protein expression and secretion levels,
we compared the cellular and secreted hIL-6-hlyAs
amounts of di¡erent hIL-6-secreting E. coli strains by
qualitative Western blot analysis. As documented in Fig.
1, E. coli (pCH1A) gave a signi¢cant immunoreactive
band, corresponding to a hIL-6HlyAs concentration of
around 25 Wg l31 culture supernatant. Upon introduction
of the hIL-6-hlyAs expression plasmid pYL3E, secreted
fusion protein amounts increased by an estimated 10-
fold. The E. coli strain carrying plasmid pYL3E only did
not reveal any signi¢cant full-length hIL-6HlyAs band,
demonstrating that the increased hIL-6-HlyAs accumula-
tion in the culture supernatant was due to Hly-mediated
secretion rather than unspeci¢c cell lysis caused by the
higher expression levels. However, extended culturing
time led to appearance of small amounts of a truncated
hIL-6HlyAs protein in both pYL3E-bearing E. coli strains.
Expectedly, the E. coli strains carrying the control plasmid
pCH2G and pACYC184 (data not shown), respectively,
did not show any immunoreactive bands in the culture
supernatants. Comparative Western blot analysis of the
corresponding cell lysates revealed a comparably strong
hIL-6-HlyAs band for both E. coli (pCH1A+pYL3E)
and E. coli (pYL3E) (data not shown). This observation
con¢rmed that only a small part of the synthesized hIL-6
fusion protein becomes actually secreted while the vast
majority of the protein remains within the cells. Our
own data and those published by other researchers sup-
FEMSIM 1522 16-6-03
139
port the assumption that the Hly secretion system func-
tions equally well in E. coli and S. typhimurium [18].
Therefore, it appeared reasonable to expect a similar in-
crease in secretory hIL-6-HlyAs expression in a S. typhi-
murium strain carrying the two-plasmid Hly expression
system (see Fig. 2B), progressing with comparative immu-
nization studies in mice.
3.2. In vitro and in vivo stability of improved
hIL-6-HlyAs -secreting strains
Comparative growth analysis over 30-h periods revealed
that the additional hIL-6-HlyAs synthesis directed by plas-
mid pYL3E did not adversely a¡ect the bacterial replica-
tion rate (data not shown). The stability of S. typhimurium
strains carrying plasmid pYL3E and the two plasmids
YL3E and pCH1A as well as the control strains was de-
termined by growth in vitro without antibiotics. X4064
maintained pYL3E with a segregation rate of less than
10% for approximately 90 generations, and thus revealed
a similar stability as that observed for pCH1A (data not
shown). A similar stability was found for the two plasmids
in X4064(pYL3E+pCH1A), indicating that homologous
recombination between the two hIL-6-hlyAs gene copies
appeared not to be a problem.
Fig. 2. In vivo stability of hIL-6-HlyAs -secreting S. typhimurium strains.
Amp- and Amp/Cm-resistant Salmonella clones recovered from organ
samples of immunized mice were analyzed for the presence of plasmids
and secretory hIL-6-HlyAs expression. A: Agarose gel (0.8%) of isolated
plasmids. The arrows mark the position of plasmid pCH1A and
pYL3E, respectively. Additional DNA bands re£ect multimeric forms of
pCH1A. Lanes: 1, 1-kb ladder standard ; 2+3, isolates from X4064-
(pCH2G)-immunized mice; 4+5, isolates from X4064(pCH1A)-immu-
nized mice; 6+7, isolates from X4064(pYL3E+pCH1A)-immunized
mice; 8, mixture of puri¢ed plasmids pYL3E and pCH1A. B: Western
blot analysis. 25-Wl volumes of stationary-phase culture supernatants of
the di¡erent Salmonella isolates were separated in a 12% SDS^PAA gel
and after transfer to nitrocellulose the membrane probed with hIL-6-
speci¢c mAb followed by chemoluminescence detection of bound anti-
bodies. The arrows indicate the full-length protein and the major degra-
dation product of hIL-6-HlyAs . Lanes: 1+2, isolates from X4064-
(pCH2G)-immunized mice; 3+4, isolates from X4064(pCH1A)-immu-
nized mice; 5+6, isolates from X4064(pYL3E+pCH1A)-immunized
mice; 7+8, mature hIL-6 standard (0.25 and 0.5 ng).
140 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145

To evaluate the in vivo stability of the strains, antibi-

otic-resistant Salmonella cells recovered from spleen and
liver of immunized mice 11 and 17 days after the second
booster immunization were analyzed for the presence and
integrity of the plasmids. As documented in Fig. 2A for
selected Salmonella clones, all analyzed colonies carried
plasmids of the appropriate sizes. The functionality of
the plasmids was con¢rmed by Western blot analysis of
the culture supernatants. HIL-6-speci¢c immunoreactive
bands corresponding to the size of hIL-6-HlyAs were
found in all these clones. Moreover, signi¢cantly higher
levels of hIL-6-HlyAs were detected in culture superna-
tants of Salmonella isolates recovered from mice immu-
nized with X4064(pYL3E+pCH1A), con¢rming the high
in vitro and in vivo stability of this strain (Fig. 2B).

3.3. Immunogenicity of secreted human IL-6 protein

To be able to di¡erentiate immunologically between re-

combinant IL-6 produced by administered Salmonella
strains and endogenous mouse IL-6 we chose the human
protein variant. Human and mouse IL-6 reveal similar
biological activity in the mouse system and therefore
should develop the same biological e¡ect. Since mouse
and human IL-6 share only 40% sequence identity, one
could expect that hIL-6 is immunogenic in mice. To ensure
that not an immune response to in situ synthesized hIL-6-
HlyAs is interfering with the expected immunomodulatory
e¡ect we measured hIL-6-speci¢c sera IgG titers in immu-
nized mice. Even 8 weeks after the primary immunization
we could not detect any signi¢cant antibody titers in mice
immunized with any of the hIL-6-producing S. typhimuri-
um strains, demonstrating that within the experimental
timeframe no immunity to recombinant IL-6 occurred
(data not shown).
3.4. E¡ect of IL-6 expression on systemic anti-Salmonella
immune responses
From previous immunization experiments with S. typh-
imurium X4064 expressing other recombinant vaccine anti-
gens we knew that in BALB/c mice maximal humoral
immune responses to Salmonella antigens required two
booster immunizations (unpublished data). Based on that
information, we designed the immunization protocol in
such a way that after primary immunization mice received
two booster immunizations in 2-week intervals before ¢nal
assessment of systemic and secretory immune responses.
To compare ¢nal immune responses to the di¡erent IL-
6-expressing Salmonella strains, we initially analyzed IgA-
and IgG-speci¢c sera titers in individual mice of di¡erent
groups. As shown in Fig. 3A, antibody titers measured
against whole-cell homogenates were relatively consistent
for the mice within the same group. There were no signi¢-
cant di¡erences in the antibody titers among the groups,
suggesting that IL-6 production had no impact on overall
Fig. 3. Ig subtype-speci¢c serum antibody levels to Salmonella cell ho-
mogenate (A) and puri¢ed LPS (B). IgA- (open symbols) and IgG-
speci¢c antibody titers (¢lled symbols) were measured 11 and 17 days,
respectively, after second booster immunization. For IgG titer measure-
ment, sera samples were diluted from 1:300 to 1:72 900 in serial one to
three dilution steps, while dilution range for IgA measurement started
at 1:100. Duplicate samples were measured and averages of four inde-
pendent titer calculations are shown for individual sera. Mice numbers
per group were six to eight.
immune responses to Salmonella carrier. We then com-
pared antibody titers to LPS as a de¢ned T-cell-indepen-
dent antigen (Fig. 3B). In contrast to sera titers to whole
Salmonella cells both LPS-speci¢c IgA and IgG titers
revealed a large mouse-to-mouse variation within each
group, indicating that some mice hyper-responded to the
LPS antigen while others developed comparably weak im-
mune responses. This extreme variation was independent
of the strain used for the immunization. Despite the large
£uctuation in individual immune responses statistical com-
parison of the titers revealed a signi¢cant di¡erence
(P 6 0.05) in the LPS-speci¢c IgA titers between the group
of mice immunized with X4064(pCH1A+pYL3E) and the
group immunized with the control strain X4064(pCH2G).
On the contrary, no statistically signi¢cant di¡erences be-
tween the groups were seen for the IgA and IgG titers
measured against whole cell homogenate.
For a more detailed comparison of the sera titers be-
tween the groups, sera samples of hyper-responding mice
were omitted from the analysis. As shown in Fig. 4, mean
IgA and IgG titers against whole Salmonella antigens in-
creased in all groups by 2^2.5-fold and about 5-fold, re-
spectively, after the two booster immunizations. No sig-
ni¢cant di¡erences were seen between the mice immunized

FEMSIM 1522 16-6-03

 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145 141

Fig. 4. Mean serum antibody titers in mice immunized with di¡erent IL-6-producing S. typhimurium X4064 strains. A: Ig subtype-speci¢c responses to
whole cell homogenate. B: Ig subtype-speci¢c responses to LPS. Titers represent the mean of sera from six mice (X4064(pCH1A+pYL3E)) and four
mice (remaining groups), respectively. Sera samples were collected prior to infection (¢lled bars), at day 26 after primary infection (hatched bars), and
at days 11 and 17, respectively, after second booster immunization (open bars). The numbers are based on duplicate sample measurements and averages
of four independent titer calculations for individual sera. Error bars indicate the S.E.M. in each group. Asterisks indicate signi¢cant di¡erences
(P 6 0.05) to the control group immunized with X4064(pCH2G) at day 26 and at the end of the experiment.

with any of the IL-6-producing Salmonella strains and the
mice immunized with the X4064(pCH2G) control strain.
Compared to the control group, LPS-speci¢c IgA re-
sponses were about four-fold higher in the mice immu-
nized with X4064(pCH1A+pYL3E) after both the primary
and the second booster immunization, suggesting that
hIL-6 secretion as well as secreted hIL-6 levels are crucial
for an immuno-adjuvant e¡ect (Fig. 4B). Mice immunized
with X4064(pCH1A) or the strain with the intracellularly
arrested hIL-6 revealed a weak increase in IgA responses,
which did not reach statistical signi¢cance due to a large
variation. LPS-speci¢c IgG titers were even further up
in mice given X4064(pCH1A+pYL3E), resulting in an
about eight-fold increase over the mean titer in the
control group. On the other hand, X4064(pCH1A)- and
X4064(pYL3E)-immunized mice revealed only about 2.5-
fold increases in their LPS-speci¢c IgG titers. Generally,
booster immunizations resulted in a signi¢cantly larger
increase in LPS-speci¢c IgG titers than in IgA titers.
3.5. Modulation of Ab-producing cell populations in spleen
and Peyer’s patches
To con¢rm the IL-6-mediated immuno-enhancing ef-
fects we additionally examined spleen and Peyer’s patches
in immunized mice after second booster immunization for
cells secreting anti-LPS antibodies. As documented in Fig.
5, a two-fold increase in the numbers of IgG-producing
cells in the mice immunized with X4064(pCH1A+pYL3E),
compared to the mice infected with X4064(pYL3E), oc-
curred. Furthermore, IgG-speci¢c ASCs were signi¢cantly
more in X4064(pCH1A+pYL3E)-immunized mice than in
mice infected with the control strain. In contrast to the
sera antibody titers, IgA-speci¢c ASC numbers were in the
same range for the mice infected with any of the IL-6-
producing strains and the control strain (Fig. 5). Com-
pared to the splenocytes, in Peyer’s patch cells IgA-speci¢c
ASCs were slightly increased over IgG-speci¢c ASCs.
Although ASC numbers for Peyer’s patches were generally

FEMSIM 1522 16-6-03

142 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
higher, no signi¢cant di¡erences were found between the
various groups for IgG and IgA (data not shown).
3.6. E¡ect of IL-6 expression on secretory anti-Salmonella
immune responses
To ensure that antibody titers measured in feces extracts
represent secreted antibodies and are not caused by blood-
borne antibody contaminations, we initially compared the
ratio of IgG and IgA levels in sera and lavage sam-
ples from mice immunized with the IL-6-negative strain
X4064(pCH2G). The average ratio of IgA to IgG was
1 to 11.5 for sera, while the ratio in gut lavages was in-
creased to 3.5 to 1.
As for the sera titers against whole Salmonella antigens,
there was no di¡erence in the mean IgG and IgA sera
titers between the groups immunized with the various
IL-6-producing strains and the X4064(pCH2G) control
strain, (data not shown). In contrast to the sera titers,
LPS-speci¢c IgA titers in gut lavages of mice given
X4064(pCH1A+pYL3E) were slightly (maximal two-fold)
increased over the mean titer measured in X4064(pCH1A)-
and X4064(pYL3E)-immunized mice (Fig. 6). Further-
more, LPS-speci¢c IgG titers were at the same level in
both groups immunized with the hIL-6-secreting strains
and were about two-fold higher than in the group given
the Salmonella strain with the intracellular arrested hIL-6.
Neither for IgA nor IgG di¡erences in the LPS-speci¢c
titers among the various groups bore a close statistical
examination due to the small sample number and the large
variance. These results suggest that under the experimental
setting the hIL-6 secretion has no impact on immune re-
Fig. 5. LPS-speci¢c ELISPOTS in spleens of immunized mice. Spleno-
cytes were prepared from animals after the second booster immunization
and 5U105 cells seeded per well in the ELISPOT assay. IgA- (¢lled
bars) and IgG-speci¢c ASCs (open bars) represent the mean ASC num-
ber from six mice (X4064(pCH1A+pYL3E)) and four mice (remaining
groups), respectively. The numbers are based on triplicate sample mea-
surements and error bars indicate the S.E.M. within each group. The as-
terisk indicates a signi¢cant di¡erence to the X4064(pCH2G)-immunized
control group (P 6 0.05).
FEMSIM 1522 16-6-03
Fig. 6. LPS-speci¢c intestinal secretory antibody responses in mice im-
munized with di¡erent IL-6-producing S. typhimurium X4064 strains.
IgA (¢lled bars) and IgG titers (open bars) represent the mean antibody
levels in fecal extracts from six mice (X4064(pCH1A+pYL3E)) and four
mice (remaining groups), respectively. For titer measurement, lavage
samples were diluted up to 1:160 in serial one to two dilution steps.
The numbers are based on duplicate sample measurements and averages
of four independent titer calculations for individual sera.
sponses triggered against LPS, neither did the amount of
secreted hIL-6 a¡ect the immune responses.
4. Discussion
The results presented demonstrate that IL-6 expressed
and secreted by Salmonella vaccine strains can enhance
systemic immunity against bacterial LPS, indicating that
Salmonella-derived recombinant IL-6 could function as an
immunotherapeutics capable of modulating induced im-
mune responses against T-cell-independent antigens. Fur-
thermore, data support the assumption that for devel-
opment of the immunomodulatory action extracellular
delivery and the amount of IL-6 are crucial. Surprisingly,
IL-6 extracellularly expressed by the Salmonella vaccine
strains a¡ected both IgG and IgA titers. In contrast to
the proposed role of IL-6 in the regulation of mucosal
IgA responses [28], we could not detect a signi¢cant e¡ect
on secretory antibody production in the gastrointestinal
tract, although titers were higher in the groups immunized
with the IL-6-expressing strains.
We have previously demonstrated that hIL-6-HlyAs
producted by S. typhimurium X4064 remains soluble and
retains full biological activity when secreted into the cul-
ture supernatant, while the intracellular non-secreted por-
tion of the fusion protein forms insoluble aggregates,
which lack bioactivity [22,23]. It is probably reasonable
to assume that we may face a similar situation in vivo.
The extracellular delivery of IL-6 is probably the reason
that, in contrast to Dunstan and coworkers [10], who ear-
lier investigated the therapeutic potential of IL-6 expressed
by Salmonella vaccine strains in a mouse immunization
model, we were able to demonstrate an immunostimulato-
ry e¡ect. Dunstan et al. used a standard plasmid expres-
sion vector for intracellular accumulation of recombinant
 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
murine IL-6 in the Salmonella strain. Although they re-
ported IL-6 proliferative activity for cell lysates as well as
for culture supernatant, the portion of biologically active
IL-6 protein, which actually became liberated from the
cells, may have been too low to trigger any in vivo e¡ect
in mice. Considering their negative results and postulating
that activity and bioavailibility of IL-6 are critical for im-
munostimulatory action, we decided to use a secretory
expression system for extracellular delivery of IL-6 by
the Salmonella carrier.
Expression of IL-4, as another Th2 cytokine [29], in
Salmonella vaccine strains revealed similar negative results
with respect to a proposed immunomodulatory function
on humoral immune responses [11]. Using a similar plas-
mid vector for IL-4 expression as Duncan et al. [10] did,
they could not detect any enhancing or inhibitory e¡ects
on Ig subtype-speci¢c immune responses either to whole
Salmonella cells or to puri¢ed LPS and £agella antigen,
respectively. Again, signi¢cant IL-4 proliferative activity
was reported for cell lysates but essentially no bioactivity
was detectable in vitro in culture supernatants, suggesting
that the bioavailibility of IL-4 in their system depends on
the portion of IL-4 protein that becomes liberated during
the desintegration of Salmonella cells. Since IL-4, like IL-
6, tends to form insoluble inclusion bodies when expressed
at high levels in bacterial systems the amount of soluble
and biologically active IL-4 protein delivered extracellu-
larly by the Salmonella cells may have been very low.
So far, IL-5 is the only Th2 cytokine that has been
shown to enhance humoral immune responses to bacterial
antigens when expressed in Salmonella dublin vaccine
strains as a fusion to thioredoxin [9]. In contrast to our
results with IL-6, IL-5 additionally stimulated mucosal
IgA responses to LPS. However, the important di¡erence
between both cytokines is that IL-5 apparently functions
as a secretory IgAþ B-cell terminal di¡erentiation factor,
not promoting an isogenic switch to IgA [30]. Also, IL-6 is
thought to control mucosal IgA responses at the post-
switch level but, in contrast to IL-5, it seems to act syner-
gically with other Th2 type cytokines [31]. Surprisingly,
however, IL-5 production in Salmonella was promoted
by the same plasmid vector [9] previously used for intra-
cellular expression of IL-4 and IL-6 cDNA, implying that
su⁄cient amounts of soluble active homodimeric protein
must have been released by the cells to trigger this e¡ect
[14].
Live Salmonella expressing a recombinant vaccine anti-
gen are promising vaccine candidates in humans and ani-
mals. However, the dominant portion of the induced im-
munity is directed towards Salmonella surface antigens
and comparable weak immune responses are induced to
recombinantly expressed vaccine antigens. One approach
by which immune responses to recombinant vaccine anti-
gens may be speci¢cally enhanced is through secretion and
surface presentation of the vaccine antigen (see [32], this
issue). Another approach to achieve a stimulation or mod-
FEMSIM 1522 16-6-03
143
ulation of immune responses is the co-delivery of immu-
nocompetent cytokines by the Salmonella vaccine strain.
While the feasibility of both approaches has been demon-
strated to some extent, the latter has the advantage that
the same cytokine-producing Salmonella strain can be uni-
versally applied as vaccine carrier for a large number of
recombinant antigens.
However, one major issue, which has to be addressed in
that context is the possible impact of the in situ cytokine
synthesis on the infectiveness and virulence of the Salmo-
nella vaccine strain. Salmonella usually stimulate protec-
tive Th1-type responses during infection, which includes
activation of macrophages, while Th2-type responses
play only a minor role in the establishment of protective
immunity to salmonellosis [33]. One may speculate that
expression of IL-6 by the Salmonella cells leads to a gen-
eral shift from Th1- to Th2-type immunity, which should
consequently result in reduced clearing rate and prolonged
colonization by this Salmonella strain, eventually a¡ecting
susceptibility of mice for that particular strain. Being
aware of that possibility, we reduced bacterial dose in
the immunization experiments from the usual 5U109 to
2U109 . So far, we did not see any marked di¡erences in
the numbers of persisting bacteria in spleens and livers of
immunized mice (data not shown). However, because of
the reduced infection dose bacterial numbers in spleen and
liver homogenates were rather low. This speculation op-
poses the results of Duncan et al. [10], who reported a
signi¢cant reduction in bacterial numbers in spleen of
mice infected with an IL-6-producing but non-secreting
Salmonella strain. On the other hand, IL-4 and IL-5 ex-
pression in Salmonella strains leads to signi¢cantly higher
in vivo survival rates in spleens and livers of immunized
mice compared to the parental strain carrying the plasmid
vector alone [9,11]. Further experimental work, including
more detailed dissection of the Salmonella-induced im-
mune responses and monitoring of the relative clearance
rates, will be necessary to gain a precise picture of that
important issue a¡ecting the development of recombinant
Salmonella-based vaccines.
At that stage, we cannot rule out that the observed im-
munostimulatory e¡ect monitored in mice immunized with
X4064(pCH1A+pYL3E) is actually due to a direct stimu-
lation of the Th2 pathway or certain B-cell populations. It
is also possible that the recombinant IL-6 secreted by the
Salmonella cells may indirectly cause the enhancement of
Ig subtype-speci¢c immune responses to LPS by modulat-
ing certain cytokine levels, thus a¡ecting the overall cyto-
kine network involved in the regulation of the Salmonella-
speci¢c immune response. To ensure that the in situ syn-
thesized IL-6 is the primary immunotherapeutic agent, im-
munization experiments in the absence of endogenous
mouse IL-6 in IL-6-de¢cient mouse strains will be neces-
sary.
To the best of our knowledge, this is the ¢rst report
demonstrating that expression of a recombinant IL-6 in
144 Y. Li et al. / FEMS Immunology and Medical Microbiology 37 (2003) 135^145
Salmonella vaccine strains can in£uence humoral re-
sponses against Salmonella antigens. Against our initial
proposal, IL-6 expression led to an increase in both IgA
and IgG antibody titers in immunized mice. So far, we
have focused on LPS as a T-cell-independent antigen. Cer-
tainly similar investigations for T-cell-dependent protein
antigens, such as £agella, will be required to further delin-
eate the immunomodulatory e¡ect of the Salmonella-
derived IL-6. Apart from that, immunization studies in
mouse strains with a di¡erent genetic background will
eventually con¢rm the immunostimulatory e¡ect caused
by the IL-6-secreting Salmonella cells, thereby supporting
the signi¢cance of these ¢ndings.
Acknowledgements
We thank W. Goebel and I. Gentschev (Theodor-Bo-
veri-Institut, Wu«rzburg, Germany) for providing DNA
and the sequence of plasmid pMOhly1, respectively. We
also thank R. Strugnell (University of Melbourne, Parks-
ville, Australia) for suggestions and helpful discussion and
G. Herger (Institut fu«r Medizinische Mikrobiologie und
Hygiene, Freiburg, Germany) for carefully reading the
manuscript. This work was in part supported by the Deut-
sche Bundeswehr and Deutsche Mucoviszidose Gesell-
schaft.
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