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The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. These results indicate that a Th1 response is sufficient to
protect against cutaneous leishmaniasis, but the induction of a
simultaneous Th2 response abrogates the Th1 effector function. DNA
vaccines may therefore have an advantage for diseases in which
protection depends on the induction of Th1 responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but no IL-4. Furthermore, there is considerable evidence that Th2-type
responses and the production of IL-4 result in the inability to control
disease, or result in disease exacerbation (2, 3). The severity of
disease in murine cutaneous leishmaniasis is better correlated with the
presence of IL-4 than the lack of production of IFN- (4, 5, 6).
A critical question therefore arises. Does protective vaccination
require not only the induction of T cells producing IFN-, but also
the prevention of a Th2-type response?
Analysis of these issues has been complicated because most immune responses involve significant contributions of both types of cytokines (7, 8, 9), and also because Th1 and Th2 responses tend to interact with each other in complex feedback loops (1). In addition, there are currently no lymphocyte surface markers that allow the physical separation of T cells secreting different types of cytokines. Accordingly, it has been difficult to dissect out the relative contribution of each T cell subset. A model system in which pure Th1 or Th2 responses are obtained would be very useful in addressing these issues.
The L. major parasite surface Ag-2 (PSA-2)3 is a family of glycosylinositol phospholipid-anchored polypeptides with approximate m.w.s of 96,000, 80,000, and 50,000 (10). A fourth member of this family with a m.w. of 50,000 is differentially expressed by L. major amastigotes (11). It has recently been demonstrated that vaccination with native PSA-2 proteins and Corynebacterium parvum as adjuvant can protect mice against infection (12). Protective vaccination was mediated by a Th1 type of immune response, as has been the case for the L. major Ags gp63 and gp46/M2 (13).
We have now exploited the PSA-2 to develop experimental systems in which we use the vaccination of mice with DNA encoding PSA-2 or with PSA-2 in immune-stimulating complexes (iscoms) (Iscom adjuvant; Iscotec AB, Uppsala, Sweden) to generate immune responses with varying degrees of Th1-like and Th2-like properties. DNA vaccines have previously been shown to preferentially induce Th1-like immune responses and have been suggested to down-regulate Th2 responses (14). Xu and Liew have shown that a plasmid encoding the leishmanial Ag gp63 induced a Th1 response in vaccinated mice (15). Recently, Gurunathan et al. (16) have shown that DNA encoding the LACK Ag induced protection in BALB/c mice. Protection seemed to be associated with IL-12 production. Iscoms are cage-like adjuvant structures that have been demonstrated to promote the development of strong Th1 responses, but also to activate Th2-like cells (17, 18).
We have used these two approaches to address the issue of the generation of protective immunity in cutaneous leishmaniasis. We show that vaccination with PSA-2 DNA can protect mice against L. major infection, and that vaccinated mice develop a virtually exclusive Th1-type of immune response. In contrast, PSA-2 iscoms induce an immune response with mixed Th1-like and Th2-like properties that is unable to confer protection. These results provide the clearest evidence to date that although a Th1 response is sufficient to cause protection against disease in experimental cutaneous leishmaniasis, this protection is abrogated if a Th2 response is simultaneously induced.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/He female mice aged 8 to 12 wk were obtained from the specific pathogen-free animal breeding facility at The Walter and Eliza Hall Institute of Medical Research (Victoria, Australia) and subsequently maintained under conventional conditions.
Parasites
The virulent cloned line V121 was derived from the Israeli L. major human isolate LRC-L137 obtained from the WHO Reference Centre for Leishmaniasis, Jerusalem, Israel. Promastigotes were maintained in vitro in Schneider’s Drosophila medium supplemented with 10% FCS (HyClone, Logan, UT) or in the biphasic blood agar (NNN) medium. The parasites and the culture conditions have been described previously (19).
Expression of rPSA-2 in transfected L. major promastigotes
The full-length L. major amastigote PSA-2 cDNA clone 2.1 (11) including the leader sequence and glycoinositol phospholipid (GPI) anchor addition sequence was cloned into the pX vector (20, 21) and expressed in L. major promastigotes. Transfected promastigotes produced this amastigote PSA-2 polypeptide in addition to the promastigote PSA-2, and both partitioned in the detergent phase during Triton X-114 phase separation, indicating that the molecule is GPI anchored (10). Expression of the amastigote PSA-2 was confirmed by Western blot analysis (data not shown).
Protein purification
Native PSA-2 polypeptides were purified from promastigote membranes by a combination of Triton X-114 solubilization-phase separation, to enrich for membrane proteins, and affinity chromatography on the mAb 11E5 as described previously (10, 11). The gene product of cDNA clone 2.1 expressed in transfected L. major promastigotes was purified from promastigote cultures by the same procedures. The purity of protein preparations was determined by SDS-PAGE and silver staining (22) and protein quantitation used the bicinchoninic acid assay (Pierce, Rockford, IL).
Expression of PSA-2 in the mammalian expression vector pCI-neo
The full-length amastigote cDNA clone described above was subcloned into the EcoRI site of the pCI-neo vector (Promega, Madison, WI) and designated membrane PSA-2 (mPSA-2). A construct encoding a water-soluble form of amastigote PSA-2 terminating immediately before the GPI anchor addition sequence was obtained from the original cDNA using the PCR and specific oligonucleotide primers. This construct was designated soluble PSA-2 (sPSA-2). COS M6 cells grown to about 50% confluence were transfected by electroporation. Briefly, 106 cells were transfected with 30 µg of DNA in 0.4-cm cuvettes (Bio-Rad, Richmond, CA) at 300 V, 125 µF using a Bio-Rad Gene Pulser. Expression of both constructs in transfected COS cells was determined by immunofluorescence using mAb 11E5 and a polyclonal rabbit serum against a carboxyl-terminal fragment of PSA-2 (23). For immunofluorescence, the transfected COS cells were grown for 48 h in DMEM with 10% FCS (DMEM/FCS) in Flaskette Chambers (Nunc, Roskilde, Denmark). Before staining, cells were fixed in 4% paraformaldehyde in PBS (pH 7.3) and permeabilized for 10 min in 0.03% saponin in PBS at 20°C. Live cells were incubated with Abs at 20°C in DMEM/FCS. PSA-2 was detected with mAb 11E5 (10 µg/ml) followed by FITC-conjugated rabbit anti-mouse Ig (Silenus, Hawthorn, Australia). Secretion of PSA-2 into cell culture supernatants was quantitated by capture ELISA using the mAb 11E5 as the capture Ab. Secreted PSA-2 was detected with biotinylated rabbit anti-PSA-2 Abs (23) followed by streptavidin conjugated to horseradish peroxidase (HRP) (Silenus).
Preparation of PSA-2 iscoms
PSA-2 iscoms were prepared using ISCOPREP 703 as a source of adjuvant-active Quillaja saponin. ISCOPREP 703 contains a mixture of two low toxicity Quillaja saponin fractions (QH-A and QH-C (24)) and was kindly provided by Iscotec, Uppsala, Sweden. PSA-2 iscoms were prepared essentially according to methods described by Lövgren and Morein (25). Briefly, 0.2 mg of a mixture of promastigote and amastigote PSA-2 was mixed with 0.2 mg of cholesterol (Sigma, St. Louis, MO) and 0.2 mg of phosphatidylcholine (Sigma) in 20% MEGA-10 detergent (Bachem, Bubendorf, Switzerland, ww/w in H2O) and 0.7 mg of ISCOPREP 703 (in H2O) in a total volume of 1 ml. The mixture was dialyzed against PBS for 24 h at 20°C and thereafter for 48 h at 4°C. The iscoms form spontaneously as the detergent is removed. Iscoms were isolated by centrifugation through a gradient of 10 to 50% sucrose at 200,000 x g for 18 h at 10°C. The protein and Quillaja saponin content of the iscoms was determined by amino acid analysis (Aminosyraanalyslaboratoriet, Uppsala, Sweden) and reverse phase HPLC (24), respectively. The cage-like morphology of PSA-2 iscoms was verified by negative staining electron microscopy (data not shown). SDS-PAGE followed by silver staining demonstrated that both promastigote PSA-2 (comprising the three polypeptides of approximate m.w.s of 96,000, 80,000, and 50,000 (10)) and the recombinant amastigote PSA-2 (a polypeptide of approximate m.w. of 40,000; 11 were efficiently incorporated into the iscoms (data not shown). Amounts of iscoms in the text refer to their protein content.
Immunizations and infection of mice
For Ab and T cell assays, groups of mice were injected twice at 4-wk intervals i.m. in both quadriceps with 20 to 50 µg of sPSA-2, mPSA-2, or control vector DNA (not containing a gene insert) or i.p. two times (at 4–6-wk intervals) with 0.5 to 1 µg of PSA-2 iscoms or iscoms and anti IL-4 mab 11B11 (American Type Culture Collection (ATCC), Rockville, MD; hybridoma line HB188). Mice were injected i.p. with 0.8 to 1 mg of anti-IL-4 mAb 11B11 together with the primary and booster iscom immunization, and 7 days before challenge infection. For analysis of serum Ab responses, mice were bled by orbital plexus puncture 14 to 21 days after the last injection. Spleens were taken at the same time points for T cell assays.
For infection, groups of six to eight mice were vaccinated with sPSA-2, mPSA-2, and vector DNA or with PSA-2 iscoms as described above except that three injections (at 4–6-wk intervals) of iscoms were given. As negative controls, mice were uninjected or injected with PBS. Two weeks after the last injection, the mice were challenged intradermally at the base of the tail with 105 live promastigotes. Lesion development was monitored weekly and assigned a score from 1 to 3 as described previously (26). Parasite burdens in lymph nodes draining the infection site were estimated 6 and 10 wk after infection by limiting dilution analysis (27).
Determination of Ab levels
Abs binding to PSA-2 were measured in individual serum samples by ELISA. Round-bottom 96-well polyvinyl chloride plates (Dynatech, Chantilly, VA) were coated with promastigote PSA-2 (1 µg/ml) for 24 h at 4°C. The plates were incubated sequentially with serially diluted sera and rabbit anti-mouse Ig conjugated to HRP (Silenus). This antiserum recognized all mouse isotypes equally (data not shown). All incubations were conducted at 20°C for 60 min. The plates were then washed four times with PBS containing 0.05% Tween-20 (PBS-T) between incubations. Sera and reagents were diluted in PBS-T containing 5% skim milk powder. The enzyme reaction was visualized by incubation with tetramethylbenzidine substrate buffer (TMB, H2O2). The reaction was stopped after 10 min by addition of 50 µl of H2SO4 and the absorbance at 450 nm was measured. For measurement of IgG isotypes, serum samples from six mice in each group were pooled. Coated plates were sequentially incubated with dilutions of pooled sera, rabbit anti-mouse IgG1 or IgG2a (Bio-Rad) and HRP-sheep anti-rabbit Ig (Bio-Rad). The remaining steps were performed as described above.
Cytokine ELISPOT assay
Single cell suspensions were prepared from spleens and 2.5
x 106 cells per well (in 0.5 ml) in 24-well plates
were incubated at 37°C and 10% CO2 with an equal volume
of PSA-2 (2 µg/ml) in DMEM containing 5% FCS and 50 µM 2-ME. Cells
primed with PSA-2 iscoms were stimulated with promastigote PSA-2. Ag
used for stimulation of cells from mice immunized with sPSA-2 DNA or
mPSA-2 DNA also included amastigote PSA-2 derived from transfected
promastigotes. Similar results were obtained with both Ag preparations
when used to stimulate spleen cells primed with either PSA-2 DNA or
PSA-2 iscoms (data not shown). Before its use in T cell assays, the
detergent in PSA-2 preparations was removed by acetone precipitation as
described (12). As a control, spleen cells were cultured without Ag.
After 72 h of incubation, the cells were washed and resuspended in
medium without Ag. In some experiments, CD4+ or
CD8+ T cells were depleted at this stage using Dynabeads
precoated with rat anti-mouse CD4 or CD8 according to the
manufacturer’s instructions (Dynal, Oslo, Norway). Depletion
efficiency was monitored by FACScan analysis and ranged from 95 to
99%. White MaxiSorp FluoroNunc 96-well plates (Nunc) were coated for
24 h at 4°C with capture Abs to IFN- (R4-6A2; ATCC) or IL-4
(11B11; ATCC) at 10 µg/ml. Plates were washed with sterile PBS and
blocked with medium. Twofold dilutions of Ag-stimulated cells (2.5
x 104 to 1 x 105 cells/well in 100 µl)
were incubated in triplicate on the plates for 20 h at 37°C, in
an atmosphere of 10% CO2. The plates were washed and
incubated for 2 h at 20°C with the detecting, biotinylated rat
anti-IFN- or IL-4 (PharMingen, San Diego, CA) diluted in PBS-T
containing 1% FCS, and for 1 h with alkaline
phosphatase-conjugated streptavidin (Silenus) diluted in PBS-T. Between
incubations, the plates were washed six times with PBS-T. Individual
cytokine-secreting spot-forming cells (SFC) were visualized and
quantitated as previously described (28). Values represent the
frequency of cytokine-secreting SFC per 106 spleen cells
and have been corrected for the frequency of SFC detected in culture
medium controls (ranging from < 5 to 43 and from 18 to 57 for SFC
producing IFN- and IL-4, respectively).
Cytokine capture ELISA
Single spleen cell suspensions (2.5 and 5 x
106 cells/ml) were stimulated with PSA-2, and culture
supernatants were collected after 48 and 96 h. The concentration
of IFN- and IL-4 in supernatants was determined as described before
(29). IL-5 was measured in the supernatants using a capture ELISA with
biotinylated anti-IL-5 Abs (PharMingen) followed by incubation with
AMDEX streptavidin conjugated to HRP (Kem-En-Tec, Melbourne,
Australia). The wells were developed for 20 min as described above for
the Ab ELISA. Values are presented as pg cytokine/ml (mean ± SD,
n = 3) and have been corrected for the level of
cytokines in culture medium controls (ranging from < 62 to 716
pg/ml of IFN- and <16 pg/ml of IL-4 and IL-5).
Statistical analysis
Significance of differences was examined using the Mann-Whitney U test. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a tool to examine the requirements for the generation of
protective immunity, we used DNA vaccination. For this purpose, the
amastigote-expressed PSA-2 gene was subcloned into the mammalian
expression vector pCI-neo to give GPI-anchored or water-soluble forms
and transfected into COS cells. Expression of both the GPI-anchored
membrane form of amastigote PSA-2 (mPSA-2) and the water-soluble form
(sPSA-2) was detected by immunofluorescence in fixed and permeabilized
cells (Fig. 1
, A and
C). The membrane-bound protein appeared abundant in
the vesicular network of the endoplasmic reticulum and the Golgi
apparatus, as well as on the cell surface. The surface location of the
protein was confirmed by immunofluorescence of live cells (Fig. 1B). In contrast, the sPSA-2, while very abundant in
the fixed and permeabilized cells, could not be detected on the surface
of living cells (Fig. 1D), consistent with it being
secreted. The presence of the protein in culture supernatants was
detected by capture ELISA (data not shown). Cells transfected with the
vector DNA alone, or untransfected cells in the transfected population,
were negative for amastigote PSA-2 (data not shown).
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Abs are believed to be of little or no importance for host
protection against L. major infection (30). However,
measurement of Ab responses can provide information concerning the
general immunogenicity of different vaccine formulations. We therefore
compared Ab responses to PSA-2 in groups of C3H/He mice immunized
twice, 4 wk apart, with 20 to 50 µg of sPSA-2, mPSA-2, or vector
control DNA or with 1 µg of PSA-2 iscoms. Mice were bled 2 wk after
the last injection and PSA-2-reactive serum Abs were measured by ELISA.
The results from one representative experiment are shown in Figure 2. Abs to PSA-2 were not detected in sera
from mice injected with vector control DNA (Fig. 2A).
Surprisingly, mPSA-2 DNA also failed to induce a detectable Ab response
(Fig. 2B), whereas sPSA-2 DNA elicited low levels of
PSA-2-specific Abs in four of seven immunized mice (Fig. 2C). Because mPSA-2 localized to the cell membrane
(Fig. 1
B), whereas sPSA-2 was secreted (Fig. 1D), it appears that PSA-2 has to be secreted from
the muscle cells to induce a B cell response. The lack of detection of
Ab responses was not due to the use of promastigote PSA-2 in the ELISA,
because similar results were obtained using recombinant amastigote
PSA-2 derived from transfected promastigotes (data not shown). In
contrast, mice injected with PSA-2 iscoms mounted a strong Ab response
(Fig. 2D) and produced Ab levels that were
approximately 2,000-fold higher than those induced by sPSA-2 DNA. As
assessed by Ab responses, PSA-2 iscoms are much more immunogenic than
the PSA-2 DNA constructs.
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The isotype profile of Ab responses depends on the cytokines
produced by Ag-specific T cells. Production of IgG2a is dependent on
IFN-, whereas IL-4 is important for the generation of high levels of
IgG1 (31). The relative production of these isotypes can thus be used
as a marker for the induction of Th1-like and Th2-like immune
responses, respectively. Analysis of pooled sera from immunized mice
revealed that sPSA-2 DNA induced Abs of the IgG2a isotype, but only
very low levels of IgG1 Abs (Fig. 3A). In contrast, PSA-2
iscoms elicited high and comparable levels of both IgG1 and IgG2a (Fig. 3B). These isotype profiles demonstrate that both
sPSA-2 DNA and PSA-2 iscoms stimulate IgG2a formation, consistent with
the induction of a Th1-type of immune response. The striking difference
in the ability to promote production of IgG1, on the other hand,
suggests that PSA-2 iscoms, but not PSA-2 DNA, induce a concomitant Th2
response.
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To determine whether the isotype profile of Abs to sPSA-2 DNA and
PSA-2 iscoms indeed reflected the activation of different T cell
subsets, we analyzed the cytokine production of spleen cells from mice
immunized with sPSA-2 DNA, mPSA-2 DNA, or PSA-2 iscoms. Control mice
were injected with vector control DNA. At 14 to 21 days after the last
injection, spleen cells were stimulated in vitro with PSA-2, and the
concentration of IL-4, IL-5, and IFN- in culture supernatants was
measured by capture ELISA. In agreement with the isotype profiles of
the Ab responses, spleen cells from mice immunized with sPSA-2 or
mPSA-2 DNA secreted low concentrations of IFN- but no detectable
IL-4 or IL-5 (Table I). PSA-2 iscoms
induced spleen cells producing high concentrations of IFN- as well
as IL-5. However, only trace levels of IL-4 were detected. Spleen cells
from control mice did not secrete detectable levels of any
cytokines.
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(IFN--SFC) or IL-4
(IL-4-SFC) in spleens from immunized mice. Spleen cells were stimulated
in vitro with PSA-2 for 72 h, and the frequency of SFC was
quantitated as described in Materials and
Methods. Consistent with the IFN- concentration in cell
culture supernatants, we found high numbers of IFN--SFC in spleens
from mice injected with PSA-2 iscoms (Fig. 4). However, PSA-2 iscoms also induced
frequencies of IL-4-SFC that were comparable with those of IFN--SFC
despite the low levels of IL-4 detected in culture supernatants. Thus,
measurement of IL-4 secretion into culture supernatants appears not to
reflect the in vivo generation of IL-4-producing cells after
immunization with PSA-2 iscoms.
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-SFC and
IL-4-SFC in spleens from mice injected with PSA-2 DNA correlated well
with the amount of cytokines detected in culture supernatants.
Immunization with sPSA-2 DNA or mPSA-2 DNA activated low but
reproducible numbers of IFN--SFC, whereas the frequency of IL-4-SFC
was below the detection limit (Fig. 4). PSA-2-specific SFC were not
detected in spleens from mice injected with vector DNA. The results
support the isotype patterns of the corresponding Ab responses, and
suggest that immunization with PSA-2 DNA induces a weak immune response
of a Th1 type. In contrast, PSA-2 iscoms strongly promote both Th1-like
and Th2-like immune responses.
IFN--producing cells in spleens from mice immunized with sPSA-2
DNA or PSA-2 iscoms are primarily CD4+ T cells
Both DNA vaccination and iscoms have been shown to efficiently
induce CD8+ CTL (34, 35). Recently, it has been
reported that murine CD8+ T cells are also able to produce
cytokines in a Th1-like and Th2-like pattern (36). Although present in
L. major-infected mice, CD8+ T cells are thought
to be of little importance in recovery from primary infection (37).
Host protection is almost completely dependent on activation of
CD4+ T cells producing Th1-like cytokines (13, 38). It was
therefore of interest to investigate whether both CD4+ and
CD8+ T cells contributed to the IFN- production induced
by PSA-2 DNA or PSA-2 iscoms. For this purpose, spleen cells from mice
immunized with sPSA-2 DNA or PSA-2 iscoms were stimulated in vitro with
PSA-2 for 72 h and subsequently depleted of either
CD4+ or CD8+ T cells. The frequency of
IFN--SFC in untreated or depleted cell samples was determined by
ELISPOT. Depletion of CD4+ T cells from spleen cells
sensitized by sPSA-2 DNA completely removed all IFN--SFC (Fig. 5). The frequency of IFN--SFC in cell
samples depleted of CD8+ T cells was similar to that of
untreated cells. In spleen cells from mice injected with PSA-2 iscoms,
depletion of CD4+ T cells resulted in an approximately 90%
reduction in the frequency of IFN--SFC (Fig. 5). After depletion of
CD8+ T cells, the number of IFN--SFC decreased by only
10%. Thus, immunization with either sPSA-2 DNA or PSA-2 iscoms
generated IFN--SFC that were predominantly CD4+ T
cells.
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Immunization with PSA-2 DNA or PSA-2 iscoms induced immune
responses that differed with respect to magnitude, Ab isotype profile,
and the pattern of cytokines produced by activated
CD4+ T cells. A critical issue in this context was
whether these responses were able to protect against L.
major infection. To determine this, groups of six to eight C3H/He
mice were vaccinated i.m. with sPSA-2 DNA or mPSA-2 DNA or i.p. with
PSA-2 iscoms. Controls were injected with vector DNA, PBS, or were
uninjected. Mice were challenged intradermally with 105
promastigotes and the course of infection was followed for 10 wk. Only
three of eight mice vaccinated with sPSA-2 DNA developed small lesions
at the site of infection, which healed after 8 wk (Fig. 6, A and B).
In the group vaccinated with mPSA-2 DNA, only two of eight mice
developed lesions (Fig. 6, A and B). All
control mice injected with PBS developed lesions that peaked in size at
about 4 to 5 wk and cured by 10 wk after infection (Fig. 6, A and B). These mice had significantly
higher lesion scores than DNA-vaccinated mice between wk 3 to 8 after
infection (p < 0.05 to 0.001). After
vaccination with vector control DNA, seven of eight mice developed
lesions (Fig. 6, A and B). The lesions
seemed to be smaller than the PBS control mice and cured in 8 rather
than 10 wk. However, in two other experiments, lesion development was
similar in mice injected with vector DNA or with PBS (data not shown).
All animals in both the DNA-vaccinated and the control group harbored
parasites in their draining lymph nodes. However, at 6 wk, a time point
close to the peak of lesion development in controls (Fig. 6, A and B), mice vaccinated with sPSA-2 or
mPSA-2 DNA had lower parasite burdens than control mice (Table II). By 10 wk postinfection, when the
lesions had cured, the difference between the groups was less
obvious.
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C; p > 0.05). All mice developed lesions
that peaked in size at about 4 to 6 wk after infection. Likewise, at 6
wk there was no apparent difference in parasite burdens in the draining
lymph nodes of mice vaccinated with PSA-2 iscoms or uninjected controls
(Table II). Lesions in vaccinated mice healed after 8 wk, whereas small
lesions were still present in some controls 10 wk after infection (Fig. 6C). In general, parasite burdens were reduced in
both groups of mice at wk 10 (Table II). This decrease was more
apparent in mice vaccinated with iscoms than in the control mice. These
results suggested that the weak but exclusive Th1 response induced by
PSA-2 DNA could protect against leishmaniasis. In contrast, a
severalfold stronger Th1 response induced by PSA-2 iscoms was unable to
protect when vaccination also induced a concomitant Th2-type of
response. To demonstrate that the lack of protection was due to the
induction of Th2 responses, and in particular IL-4, mice were injected
with anti-IL-4 mAb prior to and during vaccination with iscoms (2).
The anti-IL-4-treated mice vaccinated with PSA-2 iscoms developed
smaller lesions that cured faster than the controls (Fig. 6C). The disease pattern in these mice was similar to
that observed in the DNA-vaccinated mice (Fig. 6A).
Like immunization with DNA, injection of iscoms and anti-IL-4
induced IFN--SFC but no detectable IL-4-SFC, as measured by ELISPOT
(data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A question of considerable biologic and practical importance is whether
prophylactic vaccination must induce an exclusive Th1 response to
protect against leishmaniasis, or whether a mixed Th1/Th2 response is
sufficient. Would exposure to natural infection recall specifically the
Th1 component of the mixed response and lead to protection? Our results
suggest that this is not the case. PSA-2 iscoms induced a strong but
mixed Th1/Th2 CD4+ T cell response. Despite the activation
of large numbers of CD4+ T cells secreting IFN-, this
response did not protect from infection. In contrast to iscoms, PSA-2
DNA activated low numbers of CD4+ T cells secreting IFN-
but no detectable IL-4 or IL-5. Even though this response was weak, it
protected C3H/He mice of intermediate genetic susceptibility (40) from
infection. These data indicate that priming of a weak Th1 type of
response is sufficient for protection. However, the presence of a
concomitant Th2 response abrogates even a strong Th1 effector function,
probably due to the regulatory role of Th2-like cytokines (1). This is
further supported by our finding that administration of anti-IL-4
Abs before and during the iscom immunization led to the dampening of
the Th2 response and to concomitant protection. The generation of
protective immunity is therefore dependent on the induction of an
exclusive Th1 response and, most importantly, the activation of
Th2-like cells must be prevented.
Our observations are somewhat surprising because of evidence that
Th1-like cytokines can down-regulate Th2 responses. It is believed that
the relative levels of protective Th1-like or noncurative Th2-like
cytokines early in infection are important for the outcome of murine
leishmaniasis (13). For example, injection of the Th1 cytokine IL-12
led to the cure of leishmaniasis if administered early in disease (41).
Infection of C3H/He mice results in early production of IFN-, and
this production has been suggested to facilitate Th1 cell development
and therefore promote healing (33, 42, 43, 44). From the data presented
here, it is reasonable to assume that this primary Th1-like response to
L. major infection was boosted by the IFN--producing
CD4+ T cells that were induced by immunization with PSA-2
iscoms. However, PSA-2 iscoms also induced high numbers of
IL-4-secreting CD4+ T cells and stimulated secretion of
high concentrations of IL-5. In addition, immunization with iscoms has
been demonstrated to result in production of several other cytokines,
including IL-2, IL-10, and IL-12 (17, 45, 46, 47). Although the cytokine
response to PSA-2 iscoms may therefore have been more complex than
observed in our assays, it is likely that recall of T cells of a Th2
type by infection, possibly together with a wave of primary Th2
response, was sufficient to abrogate the protective effects of the
simultaneous production of Th1-like cytokines. In this context, the
importance of IL-4 is supported by several other studies suggesting
that susceptibility to leishmaniasis correlates with the production of
IL-4 rather than the lack of IFN- (4, 6). Moreover, BALB/c mice
deficient in IL-4 and BALB/c or C3H/He mice injected with anti-IL-4
Abs resist L. major infection (this study and Refs. 5, 48,
and 49).
In contrast to the PSA-2 iscoms, both PSA-2 DNA constructs induced only
low numbers of CD4+ T cells secreting IFN-.
Nevertheless, mice immunized with PSA-2 DNA developed significantly
smaller lesions and harbored fewer parasites in their lymph nodes than
control mice. The ability of DNA vaccination to induce protective
immunity has been demonstrated in a variety of infectious diseases.
However, most studies address the use of plasmid DNA to elicit
neutralizing Abs and to activate CD8+ CTL (50, 51).
L. major is an obligatory intracellular parasite that
propagates in macrophages. Immune recovery depends on intracellular
killing of the parasite after macrophage activation by cytokines
secreted by Th1-like cells. The ability of PSA-2 DNA to vaccinate
against leishmaniasis thus predicts a novel use of DNA vaccines: to
generate protective immunity by inducing a defined population of
CD4+ T cells to secrete Th1-like cytokines. The only other
example of DNA-induced protection in leishmaniasis is the LACK Ag.
However, surprisingly and unexpectedly, protection was dependent on the
CD8+ subset of T cells (16). The only examples of
DNA-induced CD4+ T cell-mediated protection in other
diseases are the demonstration that vaccination with DNA encoding
herpes simplex virus glycoprotein B induced CD4+ T cells
with a type 1 cytokine profile that protected mice from infection (52),
and vaccination of mice with DNA encoding mycobacterial Ags afforded
protection from challenge with Mycobacterium tuberculosis
(53, 54). Similar to leishmaniasis, Th1-like CD4+ T cells
are believed to be essential for protective immunity in these diseases
(52, 55). However, other effector mechanisms, such as CTL, are also
important (52, 56).
Generation of long-term protective immunity against leishmaniasis must
induce memory T cells, which upon encounter with the parasite are
stimulated to secrete protective Th1-like cytokines. An interesting
possibility is that the low numbers of IFN--producing T cells
detected after vaccination with PSA-2 DNA primarily reflect priming of
such memory cells, and upon natural infection this memory is recalled
with sufficient efficacy to provide host protection. Since DNA
vaccination has been demonstrated to elicit long-lasting Ab responses
(57, 58), it is likely that DNA vaccines are also able to induce T cell
responses of long duration. We are presently examining the potential of
PSA-2 DNA constructs to generate long-term protective immunity against
leishmaniasis. Data from our laboratory also indicate that injection of
the PSA-2 DNA constructs into C3H/He mice 1 or 2 wk after infection
with live L. major led to the rapid cure of lesions,
suggesting the possible use of DNA vaccines not only prophylactically,
but also therapeutically (E. Handman et al., unpublished
observations).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Emanuela Handman, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia.
3 Abbreviations used in this paper: PSA-2, parasite surface Ag-2; iscom, immune-stimulating complex; mPSA-2, membrane PSA-2; sPSA-2, soluble PSA-2; HRP, horseradish peroxidase; PBS-T, PBS containing 0.05% Tween-20; SFC, spot-forming cells; GPI, glycoinositol phospholipid; IFN--SFC, cytokine-secreting SFC producing IFN-; IL-4-SFC, cytokine-secreting SFC producing IL-4.
Received for publication March 10, 1997. Accepted for publication December 12, 1997.
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or interleukin-4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59. production correlates with the severity of disease in murine cutaneous leishmaniasis. Infect. Immun. 61:3459. secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis. J. Immunol. 149:2715.[Abstract]
that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. J. Exp. Med. 178:567. in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178:2249.This article has been cited by other articles:
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|
|
|
|
|
|
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|
|
 |
|
 A. Iwasaki The Importance of CD11b+ Dendritic Cells in CD4+ T Cell Activation In Vivo: With Help from Interleukin 1 J. Exp. Med., July 21, 2003; 198(2): 185 - 190. [Full Text] [PDF]
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|
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|
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|
|
|
|
|
|
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|
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|
|
 |
|
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|
|
|
|
 |
|
 E. Handman Leishmaniasis: Current Status of Vaccine Development Clin. Microbiol. Rev., April 1, 2001; 14(2): 229 - 243. [Abstract] [Full Text] [PDF]
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K. J. Stacey and J. M. Blackwell Immunostimulatory DNA as an Adjuvant in Vaccination against Leishmania major Infect. Immun., August 1, 1999; 67(8): 3719 - 3726. [Abstract] [Full Text] [PDF]
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