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Infect Immun, July 1998, p. 3183-3189, Vol. 66, No. 7
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mucosal Delivery of Murine Interleukin-2 (IL-2)
and IL-6 by Recombinant Strains of Lactococcus
lactis Coexpressing Antigen and Cytokine
Lothar
Steidler,1
Karen
Robinson,2
Lisa
Chamberlain,2
Karin M.
Schofield,2
Erik
Remaut,1
Richard W. F.
Le
Page,2 and
Jeremy M.
Wells2,*
Department of Molecular Biology, Flanders
Inter-University Institute for Biotechnology, and University of
Ghent, B-9000 Ghent, Belgium,1 and
Cortecs Centre for Vaccine Discovery, Department of
Pathology, University of Cambridge, Cambridge CB2 1QP, United
Kingdom2
Received 7 October 1997/Returned for modification 12 January
1998/Accepted 10 April 1998
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ABSTRACT |
Lactococcus lactis is a nonpathogenic and noncolonizing
bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can
also deliver cytokines to the immune system, we have constructed novel
constitutive expression strains of L. lactis which
accumulate a test antigen, tetanus toxin fragment C (TTFC), within the
cytoplasmic compartment and also secrete either murine interleukin-2
(IL-2) or IL-6. When mice were immunized intranasally with various
different expression strains of L. lactis, the anti-TTFC
antibody titers increased more rapidly and were substantially higher in
mice immunized with the bacterial strains which secreted IL-2 or IL-6
in addition to their production of TTFC. This adjuvant effect was lost
when the recombinant strains of L. lactis were killed by
pretreatment with mitomycin C and could therefore be attributed to the
secretion of IL-2 or IL-6 by the recombinant lactococci. These results
provide the first example of the use of a cytokine-secreting,
noninvasive experimental bacterial vaccine vector to enhance immune
responses to a coexpressed heterologous antigen and point the way to
experiments which will test the possible therapeutic efficacy of this
mode of cytokine delivery.
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INTRODUCTION |
The number of communicable diseases
which might feasibly be controlled by vaccination or treated by
immunotherapy is increasing rapidly, alongside advances in our
understanding of cellular and molecular biology as applied to the study
of infectious agents. However, virtually all of the numerous
recombinant antigen delivery systems developed to date have been
derived from attenuated pathogenic infectious agents, e.g., rationally
attenuated Salmonella spp. (23, 38) or
traditionally attenuated Mycobacterium bovis
(14).
By contrast, the use of Lactococcus lactis as a vaccine
vector is emerging as one of the most advanced prototypes of a possible new class of bacterial vaccines derived from noninvasive, nonpathogenic gram-positive bacteria (45). L. lactis is a
gram-positive bacterium which is classified as "generally regarded as
safe" following its long history of use for the production of
fermented milk products. As a gram-positive nonpathogen, its closest
functional relative is Streptococcus gordonii, with which it
shares the capacity to serve as an antigen delivery vehicle for mucosal
immunization (22). Yet unlike S. gordonii, which
is an oral commensal bacterium, L. lactis lacks any known
capacity to multiply in vivo, except in gnotobiotic mice
(15). Studies on the feeding of live lactococci to animals
and to human volunteers have shown that the passage of these bacteria
through the enteric tract is transitory, without any evidence of
colonization (15, 18).
The development of constitutive and inducible gene expression systems
for L. lactis has recently made it possible to undertake systematic investigations of the immunological activity of experimental recombinant lactococcal vaccines (46). We have been able to show that despite its lack of invasiveness, L. lactis is
able to deliver heterologous antigens to the systemic and mucosal
immune systems via mucosal routes (46). A number of antigens
of protozoal, bacterial, and viral origin have been efficiently
expressed by us in L. lactis, but most of our work to date
has focused on studies of immune responses to tetanus toxin fragment C
(TTFC) and to the partially protective 28-kDa (glutathione
S-transferase [SmGST]) immunogen from Schistosoma
mansoni (5) used as test immunogens.
Intranasal and oral immunization of mice with recombinant L. lactis expressing TTFC or SmGST elicits significant serum antibody responses against these antigens. In the case of TTFC, these responses proved to be protective against lethal challenge with 5 to 20 50%
lethal doses of tetanus toxin (25, 32). Additionally, oral
inoculation of lactococci expressing TTFC significantly but transiently
elevated the levels of anti-TTFC immunoglobulin A (IgA) antibodies
detected in the gut secretions (32).
In the light of our previous results, the present study was carried out
to determine whether lactococci can deliver biologically active
molecules such as cytokines as well as heterologous antigens to the
immune system. Cytokines produced by subpopulations of T cells
critically influence the balance between humoral and cell-mediated types of immune responses and are potentially useful as immune response
modulators for vaccines and immunotherapeutic agents (40).
Recombinant strains of M. bovis BCG secreting functional mammalian cytokines have been shown to be more potent stimulators of
cell-mediated immune responses than their nonrecombinant counterparts in mouse models of experimental infection (24). By contrast, antibody responses to whole bacterial cells, outer membrane proteins, or lipopolysaccharide antigens of attenuated Salmonella
typhimurium were not augmented when these strains were engineered
to express interleukin-6 (IL-6), IL-1, or IL-4 intracellularly (3,
7, 11). The influence of these cytokines on responses to
heterologous antigens expressed by these bacteria has not subsequently
been investigated. In viral vector systems, the coexpression of IL-6 has been shown to augment both systemic and mucosal antibody responses to the viral antigens (21, 30). In this study, murine IL-2 and IL-6 were chosen for expression in L. lactis, as they
have been shown to enhance antibody titers to either vaccine antigens or inactivated viral vaccines when parenterally administered either exogenously or in liposomal formulations (10, 16, 20, 35). Studies both in vitro and in vivo have suggested a potential
application for IL-6 in augmenting IgA antibody responses in mucosal B
cells or at mucosal surfaces (26, 27, 30, 33). Additionally, it has been shown that serum levels of IL-6 correlate with the serum
concentration of IgA in patients with alcoholic liver cirrhosis and
children infected with human immunodeficiency virus (8, 31),
implicating its role in augmenting IgA synthesis in the serum.
Inclusion of IL-2 in liposomes containing bacterial polysaccharide has
also been shown to enhance antibody titers of polysaccharide-specific secretory IgA and to increase the numbers of polysaccharide-specific pulmonary plasma cells more than 80-fold following intranasal immunization of mice (1).
In this report, we present results which indicate that it is possible
to prepare genetic constructs in L. lactis which confer on
this organism the capacity to deliver physiologically active quantities
of murine IL-2 and IL-6 in vivo.
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MATERIALS AND METHODS |
Recombinant DNA techniques.
PCR amplification of DNA was
performed with Vent polymerase and using conditions recommended by the
manufacturer. DNA-modifying enzymes and restriction endonucleases were
used under standard conditions and in the buffers recommended by the
manufacturers. General molecular cloning techniques and the
electrophoresis of DNA and proteins were carried out essentially as
described previously (34). L. lactis was
transformed by electroporation of cells grown in the presence of
glycine (47), and Escherichia coli was
transformed by the electroporation method of Dower et al. (9).
Fractionation of lactococci and immunoblotting.
Total-cell
protein extracts of L. lactis cells were prepared by the
method of Wells et al. (48). To recover proteins from the
cell wall of lactococci, the cell wall was enzymatically digested with
mutanolysin and lysozyme in the presence of an osmotically stabilizing
buffer. Bacteria (approximately 2.5 × 109 CFU) were
pelleted by centrifugation, washed three times in Tris-buffered saline
(0.15 M NaCl, 0.02 M Tris-HCl [pH 7.5]), resuspended in 200 µl of
20 mM Tris-HCl (pH 7.5)-10% (wt/vol) sucrose containing mutanolysin
(100 U/ml) and lysozyme (5 mg/ml), and incubated for 1 h at
37°C. After treatment with mutanolysin and lysozyme, the cells were
separated from enzymatically released cell wall material by
centrifugation. Enzymatically digested cell wall fractions and cell
pellets were boiled for 3 min in Laemmli sample buffer. Proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and electroblotted onto nitrocellulose. The transfer of protein was
checked by reversibly staining the filter with Ponceau S, after which
TTFC and or murine cytokines were detected by immunoblotting as
previously described (36, 48).
Preparation of bacterial cells for immunization.
L.
lactis strains carrying pTREX1, pT1TT, pT1TT-IL2, and pT1TT-IL6
were cultured at 30°C in M17 broth (Difco Ltd.) supplemented with
0.5% (wt/vol) glucose and 5 µg of erythromycin per ml to mid-logarithmic growth phase. The cells were washed with sterile phosphate-buffered saline (PBS) before resuspension in a solution of
0.2 M sodium bicarbonate (Sigma, Poole, Dorset, United Kingdom), 5%
casein hydrolysate [GIBCO Ltd., Paisley, United Kingdom), and 0.5%
(wt/vol) glucose (Sigma) at 5 × 1010 CFU/ml.
Mitomycin C pretreatment of bacteria.
Cells from a fresh
overnight culture at 2 × 108 CFU/ml were treated with
50 µg of mitomycin C (Sigma) per ml for 2 h at 30°C. Aliquots
of 1 ml were taken after incubation, and the concentration of cells was
determined by nephelometry. The cells were washed three times in
sterile PBS before spreading over GM17 agar plates containing the
relevant selective antibiotics in order to calculate the efficiency of
killing. In each case, fewer than 1 in 104 cells remained
viable after treatment with mitomycin C. All cells used for
immunization were washed three times in a large excess of PBS before
resuspension at 5 × 1010 CFU/ml as described above.
Immunization protocol.
Groups of six specific-pathogen-free
female C57BL/6 mice (Harlan UK Ltd. Bicester, Oxon, United Kingdom),
aged 6 to 8 weeks at day 0, were immunized intranasally with
recombinant L. lactis. Intranasal doses of 109
cells in 20 µl were applied to lightly anesthetized animals, using a
micropipette. Serum samples were taken at intervals of 14 days and were
stored at 
20°C until required.
ELISA for detection of TTFC-specific serum antibody.
Using a
method based on that of Wells et al. (48), enzyme-linked
immunosorbent assay (ELISA) plates were coated overnight at 4°C with
recombinant purified TTFC (50 ng/well; Boehringer Mannheim, East
Sussex, United Kingdom) in carbonate-bicarbonate buffer (pH 9.6). Wells
were blocked for 1 h at room temperature, using 3% bovine serum
albumin (BSA) (Sigma). Primary antisera were tested in duplicate wells,
using a twofold dilution series, including replicate wells of a
1/50-diluted preimmune serum on every plate. After 90 min, secondary
anti-mouse immunoglobulin-alkaline phosphatase conjugates (Southern
Biotechnology Associates, Inc., Birmingham, Ala.) were applied before
development, using n-nitrophenyl phosphate (Sigma) as the
substrate. Dilution curves were drawn for each sample, and the endpoint
titer was calculated as the dilution producing the same optical density
as the 1/50 dilution of a pooled preimmune serum. Statistical
comparisons between groups were made by the Mann-Whitney U test. A
P value of >0.05 was considered nonsignificant.
ELISA for detection of antilactococcal serum antibody.
An
extract of soluble lactococcal protein was prepared from the L. lactis control strain carrying pTREX1 as previously described (48). The protein concentration of the cell extract was
determined by using a Bradford assay (Bio-Rad Laboratories,
Hertfordshire, United Kingdom). ELISA plates were coated overnight with
protein extract in carbonate-bicarbonate buffer (pH 9.6) (50 ng/well) and then blocked for 1 h at room temperature with 3% BSA (Sigma). Dilutions of the primary antisera were tested in duplicate for antilactococcal antibody as described above for the TTFC ELISA.
Assay of IgA in fecal material.
Fresh fecal pellets were
collected from each group of mice and frozen at 20°C until the end
of the experiment. Soluble fecal extracts were prepared in 2-ml
microcentrifuge tubes by adding 1 ml of PBS containing 1% BSA and 1 mM
freshly added phenylmethylsulfonyl fluoride per 0.1 mg of fecal
material. The tubes were incubated overnight at 4°C to soften the
pellets and then vigorously mixed by vortexing to disrupt and suspend
all solid matter. The samples were then centrifuged at full speed in a
microcentrifuge for 5 min to pellet the insoluble material. The
supernatants were removed and assayed for total IgA concentration,
using a commercially available radial immunodiffusion test kit (The
Binding Site, Birmingham, United Kingdom) according to the
manufacturer's instructions.
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RESULTS |
Coexpression of TTFC and murine IL-2 or IL-6 in L. lactis.
To coexpress the test antigen (TTFC) together with a
cytokine, artificial operons were constructed within the lactococcal expression plasmid pTREX1 (Fig. 1)
(46). This was achieved by modifying a strain which
expresses and accumulates the model immunogen TTFC intracellularly so
that it could additionally coexpress and secrete murine IL-2 or IL-6.
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FIG. 1.
Schematic representation of pTREX1 (46) and
the expression plasmids constructed for use in L. lactis.
The location of unique restriction endonuclease sites, promoter (p1),
Shine-Dalgarno motif (SD), translation initiation start codon (ATG),
and transcription terminator
()
present in the expression cassette of pTREX1 are indicated. The
nucleotides encoding TTFC are numbered as previously described
(12). In plasmids pT1TT-IL2, pT1TT-IL6, pT1-IL2, and
pT1-IL6, the cDNA fragments encoding mature forms of murine IL-2 and
IL-6 (nucleotide numbering as in references 6 and
17) were fused to the secretion leader of L. lactis usp45 (nucleotide numbering as in reference
42).
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Briefly, the DNA fragment encoding TTFC was amplified by PCR and cloned
between blunted
SphI and
BamHI sites in pTREX1 to
generate plasmid pT1TT, which expressed TTFC intracellularly in
L. lactis. To obtain secretion of the mammalian cytokines by
L. lactis, the eukaryotic secretion signal sequences present
in the
cDNAs of IL-2 and IL-6 were replaced with the prokaryotic signal
sequence of the lactococcal
usp45 gene. Previous studies had
shown
that such fusions resulted in the secretion of fully active
cytokines
into
L. lactis culture supernatants (
36,
37). Initially the
DNA fragments encoding the mature processed
form of murine IL-2
or IL-6 were cloned into the lactococcal expression
plasmid pLET2N
(
25). DNA fragments encoding the lactococcal
usp45 secretion
signal-cytokine fusion product and its
upstream Shine-Dalgarno
sequence were then cloned downstream of the
TTFC gene in pT1TT
to generate pT1TT-IL2 and pT1TT-IL6 (Fig.
1). For
use as controls,
we constructed two strains of
L. lactis
which expressed only IL-2
or IL-6. These were constructed by cloning
PCR-amplified fragments
encoding the
L. lactis usp45
secretion signal-cytokine fusion
products and their upstream
translation initiation region (initially
constructed in pLET-based
plasmids) between the
BamHI and
BglII
sites in
pTREX1 (Fig.
1). The characteristics of the various different
strains
of recombinant
L. lactis constructed are shown in Table
1.
Immunoblotting of equal amounts of cell extract prepared from
L. lactis strains carrying plasmid pT1TT, pT1TT-IL2, or pT1TT-IL6
revealed that all three strains produced similar amounts of TTFC
(Fig.
2A). Cytokine bioassays carried out on
culture supernatants
harvested from exponentially growing cultures
showed that biologically
active IL-2 and IL-6 were secreted into the
growth medium at levels
of approximately 0.9 µg/ml by the
L. lactis strains harboring
pT1TT-IL2 and pT1TT-IL6,
respectively (Fig.
2B). Immunoblotting
with anti-IL-2 and anti-IL-6
antisera revealed that small quantities
of IL-2 and IL-6 were present
in total cell extracts prepared
from the strains carrying plasmids
pT1TT-IL2 and pT1TT-IL-6, respectively
(not shown). The IL-2 and IL-6
detected in these total cell extracts
were probably in their
unprocessed forms (signal sequence plus
cytokine) since the proteins
detected were consistently found
to be larger than purified IL-2 and
IL-6, respectively. The mature
processed forms of these cytokines were
not detected in whole
bacterial cell extracts or in enzymatically
degraded cell wall
fractions of protoplasted bacteria, indicating that
cytokines
secreted by recombinant strains of
L. lactis were
not being trapped
in the cell wall.
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FIG. 2.
(A) Immunoblotting of whole-cell extracts from
approximately 5 × 108 CFU of L. lactis
MG1363 transformed with plasmid pTREX1, pT1TT, pT1TT-IL2, or pT1TT-IL6
(as indicated) with rabbit polyclonal antiserum to TTFC. (B) Amount of
biologically active IL-2 or IL-6 detected in the culture supernatant of
the L. lactis strains based on the mean values of four
separate titrations as described previously for IL-2 and IL-6 (13,
44).
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Immunization of mice with live recombinant strains of
L. lactis secreting IL-2 or IL-6 potentiated antibody
responses to the coexpressed antigen TTFC.
Groups of six C57BL/6
mice were immunized intranasally with three doses of 109
CFU of the various strains of recombinant lactococci on days 0, 14, and
28. The TTFC-specific serum antibody titers reached mean endpoint
titers of 104 to 105 in all mice which had been
immunized with lactococcal strains expressing TTFC compared to mean
endpoint titers of approximately 50 for naive mice and mice inoculated
with a nonexpressor control strain carrying only the expression vector
pTREX1. Strikingly, the anti-TTFC antibody titers increased more
rapidly and were substantially (10- to 15-fold by day 35) higher in the
groups of mice immunized with the lactococcal strains coexpressing IL-2 (P = 0.001) or IL-6 (P = 0.001) than in
the group immunized with the strain expressing only TTFC (Fig.
3). On day 80 following immunization, the
mean TTFC antibody titers were still significantly higher in the groups
of mice immunized with the recombinant L. lactis expressing
IL-2 (P = 0.040) or IL-6 (P = 0.001)
than in the group immunized with L. lactis expressing only
TTFC.
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FIG. 3.
Mean serum anti-TTFC IgG levels of groups of six mice
intranasally inoculated on days 0, 14, and 28 with 109
recombinant L. lactis as follows:  , expressing TTFC
(pT1TT);  , expressing TTFC and cosecreting IL-2 (pT1TT-IL2);  ,
expressing TTFC and cosecreting IL-6 (pT1TT-IL6);  ,
control nonexpressor strain (pTREX1). Sera from a naive, nonvaccinated
control group ( ) were also assayed. The endpoint titer was
calculated as the dilution of serum producing the same optical density
as a 1/50 dilution of a pooled preimmune serum included in six
replicate wells on each plate. *, the endpoint titer was
significantly higher than those of the group inoculated with the pT1TT
strain (P > 0.05).
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Both IgG1 and IgG2a subclasses of anti-TTFC antibody were found in the
sera of mice immunized with TTFC-expressing lactococci.
The relative
proportions of these antibody subclass-specific responses
were not
significantly different in mice inoculated with the cytokine-secreting
lactococci. As in previous studies (
32), the serum antibody
responses directed against antigens of
L. lactis were low
(maximum
endpoint titers between 50 and 500) in comparison with the
anti-TTFC
responses (Fig.
4).
Interestingly, antibody responses directed
against antigenic components
of
L. lactis were not detectably
raised by coexpression of
the cytokines. The substantial potentiation
of the anti-TTFC response
in mice inoculated with the IL-2- and
IL-6-secreting lactococci
appeared to be restricted to the expressed
heterologous protein.
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FIG. 4.
Antilactococcal serum immunoglobulin responses of groups
of six mice intranasally inoculated on days 0, 14, and 28 ()
with 109 recombinant L. lactis as follows:  ,
expressing TTFC (pT1TT);  , expressing TTFC and cosecreting IL-2
(pT1TT-IL2);  , expressing TTFC and cosecreting IL-6 (pT1TT-IL6);
, control nonexpressor strain (pTREX1). Sera from a naive,
nonvaccinated group ( ) were also assayed. The endpoint titer was
calculated as the dilution of serum producing the same optical density
as a 1/50 dilution of a pooled preimmune serum included in six
replicate wells on each plate.
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Influence of cytokine coexpression by recombinant L. lactis on IgA responses.
To determine whether immunization
with recombinant lactococci coexpressing IL-6 or IL-2 would
specifically enhance IgA responses, we assayed for TTFC-specific IgA in
the pooled serum from groups of animals immunized intranasally with the
different strains of L. lactis (Table 1). Mice immunized
with lactococci coexpressing IL-6 had serum IgA antibodies to TTFC
substantially higher than those of the naive nonimmunized group of
animals (Fig. 5). To determine whether
the cytokines secreted by recombinant L. lactis would also
influence the production of IgA in mucosal tissues, the feces collected
from groups of mice immunized intranasally with different doses of
recombinant L. lactis and a naive control group of mice were
assayed for total IgA content by using a commercially available radial
immunodiffusion kit. At 35 days postimmunization (7 days after the
final boost), the concentrations of IgA measured in the soluble
extracts prepared from fecal material were similar for both immunized
and naive groups of mice (results not shown). These results suggest
that intranasal immunization with IL-6- or IL-2-secreting L. lactis had no measurable effect on total IgA production in the
gastrointestinal tract.
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FIG. 5.
Anti-TTFC IgA levels in pooled sera from groups of six
mice intranasally inoculated on days 0, 14, and 28 with 109
recombinant L. lactis as follows: , expressing TTFC
(pT1TT); , expressing TTFC and cosecreting IL-2 (pT1TT-IL2); ,
expressing TTFC and cosecreting IL-6 (pT1TT-IL6); ,
control nonexpressor strain (pTREX1). Sera from a naive, nonvaccinated
control group () were also assayed. The endpoint titer in the ELISA
was calculated as the dilution of serum producing the same optical
density as a 1/50 dilution of a pooled preimmune serum included in six
replicate wells on each plate.
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Influence of lactococcal cell viability on immune responses
elicited by recombinant L. lactis.
Groups of six mice were
immunized with live or mitomycin C-killed strains of L. lactis to determine whether effective cytokine delivery, as
determined by the adjuvant effect of the anti-TTFC antibody response,
was dependent on the production of active cytokine in vivo (rather than
being due to the possible release of preformed, bacterially associated
cytokine in the animal). All mice immunized with the TTFC-expressing
strains of L. lactis elicited serum antibody responses to
TTFC with peak endpoint titers of 2 × 103 to 3 × 104, compared to mean endpoint titers of approximately
50 for naive mice and control mice inoculated with a nonexpressor
control strain (Fig. 6). The mean
antibody titers elicited by live and killed L. lactis
expressing only TTFC were not significantly different on any of the
days on which serum was sampled (Fig. 6), showing (as have our previous
results [25, 32]) that antigen expression in vivo is
not required to elicit an immune response to TTFC expressed intracellularly in lactococci. However, when the cytokine-secreting strains of L. lactis were treated with mitomycin C, the
titers of anti-TTFC antibodies were not significantly different from those elicited by bacteria expressing TTFC alone, indicating that cytokine-secreting strains of lactococci need to be viable in vivo for
effective cytokine delivery to occur (Fig. 6).
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FIG. 6.
Influence of lactococcal cell viability on the immune
responses elicited by IL-2-secreting (a) and IL-6-secreting
(b) L. lactis. Shown are mean anti-TTFC serum
antibody responses of groups of six mice following intranasal
administration of 109 live (closed symbols) or mitomycin
C-killed (open symbols) recombinant L. lactis strains on
days 0, 14, and 28 () as
follows: , expressing TTFC (pT1TT); , expressing TTFC and
cosecreting IL-2 (pT1TT-IL2); , expressing TTFC and cosecreting IL-6
(pT1TT-IL6); , control nonexpressor strain (pTREX1);  , secreting
IL-2 (pT1-IL2);  , secreting IL-6 (pT1-IL6). Sera from a naive,
nonvaccinated group () were also assayed. The endpoint titer was
calculated as the dilution of serum producing the same optical density
as a 1/50 dilution of a pooled preimmune serum included in six
replicate wells on each plate.
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DISCUSSION |
The results presented here indicate that it is possible to
construct physiologically active antigen- and cytokine-expressing recombinant strains of L. lactis and that these strains
appear to constitute a simple and effective means of delivering murine IL-2 and murine IL-6 across mucosal surfaces to the immune system.
This study was made possible by constructing an artificial bicistronic
operon within the constitutive expression plasmid pTREX1 to yield
strains of L. lactis which secrete IL-2 or IL-6 and also coexpress TTFC and accumulate this protein intracellularly. These bacteria secreted biologically active IL-2 or IL-6 and elicited significantly (10- to 15-fold) higher TTFC antibody titers than did the
strains expressing only TTFC (Fig. 3). As the amounts of TTFC produced
by the different strains were similar, and since we have found
(unpublished results) that the TTFC antibody titers elicited by
recombinant strains of L. lactis expressing 10-fold-higher levels of TTFC antigen are not substantially different, the enhanced anti-TTFC immune responses must be directly attributable to the coexpression of IL-2 or IL-6.
The influence of cytokine coexpression by recombinant lactococci on IgA
responses was investigated by measuring the serum levels of anti-TTFC
IgA and total IgA recovered in the feces of mice immunized with the
various strains of L. lactis. Elevated serum anti-TTFC IgA
responses were seen only in mice immunized with recombinant lactococci
secreting IL-6. These TTFC-specific IgA serum responses were elevated
even after the primary dose but increased over time for at least 80 days postimmunization (Fig. 5). Although the differences between the
IgA levels measured in the IL-6 group of animals and those of the naive
and control groups were small (up to twofold), these differences are
significant since we have never previously been able to measure serum
IgA responses to TTFC following immunization with recombinant
TTFC-expressing lactococci which differed from those of the naive or
nonexpressor control groups. The amounts of total IgA measured in fecal
material were not increased in the groups immunized with the
cytokine-secreting strains of L. lactis. However, further
experiments aimed at measuring IgA levels in mucosal secretions at
various times after immunization with cytokine-secreting L. lactis by different routes will be needed before any firm
conclusions can be drawn as to their possible effects on IgA production
in mucosal tissues.
By comparison with the marked enhancement of the serum anti-TTFC IgG
antibody titers seen here, it was striking that the antibody responses
to lactococci were not elevated in mice inoculated with the strains
coexpressing IL-2 or IL-6 (maximum ELISA titers of 400 in all groups of
mice [Fig. 4]). We have previously observed that lactococci appear to
have a low innate antigenicity. This property may be advantageous if
the use of repeated administration of recombinant lactococci is
required.
The mode of action of the cytokine-secreting strains on the immune
system requires further investigation. Lactococci are sufficiently small (about 1 µm in diameter) to be taken up by microfold cells present in the follicular epithelium overlaying the mucosa-associated lymphoid tissue. We have shown in this report and previously that these
bacteria resemble inert microparticles to the extent that effective
presentation of TTFC by lactococci to the immune system does not
require bacterial viability (25, 32). By contrast, the
adjuvant effect on the immune responses to TTFC observed with the live
cytokine-secreting strains was lost when the bacteria were treated with
lethal doses of mitomycin C prior to immunization (Fig. 6). This result
constitutes the most direct evidence that lactococci can deliver
cytokines in vivo and that the adjuvant effect of cytokine coexpression
and secretion by lactococci is due to a level of continued
physiological activity by the lactococci in vivo. An alternative
explanation would be that the adjuvant effects observed here were due
to the release of preformed, bacterially associated cytokines by live
but not by mitomycin-killed L. lactis. We cannot completely
preclude this possibility but consider it unlikely, in view of the fact
that we could not detect processed cytokines in the digested cell wall
fractions of live protoplasted cytokine-expressing bacteria.
The finding that L. lactis can be used to deliver
biologically active cytokines to the immune system in vivo is
surprising because L. lactis is not a commensal bacterium
and is not known to replicate in vivo. The fate of intranasally
inoculated L. lactis is also not known, although it is
likely that a proportion of any inoculum given intranasally may be
inhaled and then enter the lungs or be swallowed and enter the
gastrointestinal tract. Physiological studies of the murine
nasal-associated mucosal immune system indicate that antigens and
pathogens encountered at the nasal epithelium will be sampled by
microfold cells and transported to the underlying lymphoid tissue,
where they can induce an immune response (2, 19). Here the
local production of IL-2 or IL-6 by L. lactis may stimulate
early proliferation of mucosal T cells or augment B-cell growth and/or
differentiation and immunoglobulin production (5, 28, 39, 41,
43). The results presented here suggest that it may be possible
to tailor the type of immune response elicited to antigens delivered by
lactococci through the expression of appropriate cytokines and in such
way lead to an appropriate vaccination strategy against a particular
pathogen. The striking fact that a pulse of cytokines can be delivered
by recombinant lactococci in this way also raises interesting and testable questions concerning the possible therapeutic uses of such
recombinant bacteria.
| |
ACKNOWLEDGMENTS |
We thank the Underwood Fund of the Biotechnology and Biological
Sciences Research Council (BBSRC) for providing a travel and subsistence grant to L.S. This work was supported by grants from the
BBSRC to J.M.W., K.M.S., and R.L.P., The Wellcome Trust (to K.R.), and
the EU (to L.C.). J.M.W. also gratefully acknowledges the award of an
advanced research fellowship from the BBSRC.
We thank J. Halpern for providing the gene encoding TTFC.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cortecs Centre
for Vaccine Discovery, Department of Pathology, University of
Cambridge, Tennis Court Rd., Cambridge CB2 1QP, United Kingdom. Phone:
44 1223 330245. Fax: 44 1223 333346. E-mail:
jmw16{at}mole.bio.cam.ac.uk.
Editor: V. A. Fischetti
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Infect Immun, July 1998, p. 3183-3189, Vol. 66, No. 7
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
