By
From the * Lymphokine Regulation Unit, Laboratory of Clinical Investigation, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; Laboratory of Parasitic Diseases, National Institute for Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892; § Department of Medicine, Committee on
Immunology and Gwen Kanpp Center for Lupus and Immunology Research, The University of
Chicago, Chicago, Illinois 60637; and the
Institut de Pharmacologie Moléculaire et Cellulaire,
UPR411 Centre Nationale de la Recherche Scientifique, 06560 Valbonne, France
To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted
into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible
BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with
that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK
protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred
by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic
protection was achieved by LACK DNA vaccination. The control of disease progression and
parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon- (IFN-
) production. Moreover, both the enhancement of IFN-
production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection
also abolished the protective response induced by LACK DNA vaccination, suggesting a role
for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization
may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.
Immunization with plasmid DNA has been shown to induce protective immunity in a variety of experimental
models of infection (1) through both MHC class I- (13-
15) and class II-restricted T cell responses (16). The mechanism by which DNA vaccination is able to generate these
potent immune responses appears to be through the induction of various proinflammatory cytokines elicited in response to certain immunostimulatory sequences (ISS)1 contained in the bacterial plasmid (17). In addition, recent evidence suggests that the route by which DNA is administered plays an important role in determining the type of
CD4+ T cell (i.e., Th1 or Th2 type) response generated
(18, 19). Thus, DNA vaccination can provide a useful and
an effective way in providing effective immunity to particular pathogens depending on the type of immunity required for protection.
In susceptible BALB/c mice infected with Leishmania major, the preferential expansion of parasite specific Th2-type
cells, and the relative absence of IFN- In the present study, we investigated whether DNA vaccination could be used to induce a protective response in
BALB/c mice infected with L. major. For this purpose, we
used DNA encoding the recently identified LACK antigen
(29). This 36-kD protein is a highly conserved protein
among related leishmania species and is expressed in both
promastigote and amastigote forms of the parasite. LACK
has been found to be the focus of the early immune response directed to the parasite with most LACK-reactive T
cells producing IL-4, but not IFN- Mice.
Female BALB/c mice were purchased from the Division of Cancer Treatment, National Cancer Institute (Frederick,
MD) and kept in the National Institute of Allergy and Infectious
Diseases Animal Care Facility under pathogen-free conditions.
Mice used were between 6-8 wk of age.
Media and Reagents.
HBSS (Biofluids, Rockville, MD) was used
as wash medium. RPMI-1640 (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum (Biofluids, Rockville,
MD), penicillin (100 U/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and 2-ME (0.005 mM) was used to culture splenocytes and lymph node cells. LACK recombinant protein was purified from bacteria as previously described (29).
Plasmid Construction and Purification.
A cDNA encoding a truncated LACK protein (amino acids 143-312) was cloned in frame
downstream to a Kozak consensus sequence and an initiation
codon into a pECE vector (29). The insert was excised using
HindIII and ligated into an expression vector PcDNA-3 downstream to the CMV promoter (Invitrogen, San Diego, CA). Eukaryotic expression vectors carrying cytokine cDNAs (IL-12, IL-4)
were provided by J. Haynes (Auragen, Inc., Middleton, WI). Plasmid DNA was purified by double banding cesium chloride
gradient ultracentrifugation. The 260/280 ratios ranged from 1.8 to 2.0.
Immunization.
Female BALB/c mice were injected in their
hind footpads with 100 µg of plasmid DNA suspended in 50 µl
of sterile PBS. In some experiments, 100 µg cytokine of cDNA
was combined with 100 µg LACK DNA and injected as above.
For immunization with protein, mice were injected as above
with 50 µg of recombinant LACK protein with or without 1 mg
of rIL-12 (Genetics Institute, Cambridge, MA). Mice were
boosted 2 wk later with their initial regimen.
Infectious Challenge.
L. major (WHOM/IR/ Parasite Quantitation.
Parasite loads in footpads were determined by sequential immersion of footpads in Wescodyne solution (Amsco, Erie, PA), 70% EtOH, and sterile dH2O, before homogenization of weighed tissue in microfuge tubes containing
100 µl of M199/S. Each tissue homogenate was serially diluted in
a 96-well flat-bottomed microtiter plate containing biphasic medium, prepared using 50 µl NNN medium with 30% defibrinated rabbit blood, and overlaid with 50 µl M199/S. The number of viable parasites per milligram of tissue was determined from
the highest dilution at which promastigotes could be grown out
following up to 7 d incubation at 26°C. An identical limiting dilution assay was used to quantitate viable parasites in single cell
suspensions of draining lymph nodes obtained at various times after infection.
Treatment of Mice with Neutralizing Antibodies.
Purified neutralizing mAb (1 mg/mouse intraperitoneally) against murine IL-12
(hybridoma c17.8), was obtained from Dr. G. Trinchieri (Wistar
Institute, Philadelphia, PA) and injected into BALB/c mice at the
time of initial vaccination and then on a weekly basis until 4 wk
after infection. Anti-murine CD8 (2.43) was injected into BALB/c
mice either at the time of vaccination or infection and then on a
weekly basis to ensure continuing depletion. CD8 T cell depletion was confirmed by FACS® analysis of splenocytes from euthanized mice.
Cytokine Production Assay.
At various times points after infection or immediately before infection, mice were killed and the
draining lymph nodes were harvested. Single cell preparations
from the lymph nodes were plated in triplicate in a 96-well microtiter plate at 3 × 105 cells/200 µl. LACK protein (10 µg/ml)
or media were added to cultures and supernatants were collected
48 h later and stored at Measurement of Cytokine Production.
Measurement of IFN- Measurement of Lack-specific Antibody Responses.
Pooled serum
samples (n = 7-12 mice/group) were obtained at various time
points after infection and analyzed for the presence of LACK-specific antibodies. In brief, 96-well Immulon-4 plates were coated
with LACK protein (10 µg/ml) overnight at 4°C. Plates were
blocked with 2% BSA/PBS at 37°C for 1 h to prevent nonspecific binding. Sera was added at serial fivefold dilutions (starting at
1:5) and incubated overnight at room temperature. Horseradish peroxidase-conjugated-goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Inc., Birmingham, AL) was added
for 1 h at 37°C. ABTS peroxidase substrate (KP Laboratories,
Gaithersburg, MD) was added and absorbance was read on ELISA
plate reader (Dynatech, Chantilly, VA) using a 410-nm filter referenced to 510 nm.
In the initial
experiment, the ability of LACK DNA vaccination to induce a protective response was directly compared with that
achieved by LACK protein and rIL-12. This latter immunization regimen has been previously shown to be an effective vaccine against L. major infection (29). As shown in
Fig. 1, mice vaccinated with either LACK DNA alone, or
with LACK protein and rIL-12, effectively controlled disease at 12 wk after infection compared with mice vaccinated with LACK protein alone or control DNA. Although in this particular experiment there is a modest
decrease in footpad swelling from mice vaccinated with
LACK protein and rIL-12 compared with LACK DNA
alone; this did not correlate with a reduction in parasite
load (see below). Moreover, in other experiments not shown, mice vaccinated with LACK DNA and LACK
protein plus IL-12 had similar degrees of footpad swelling.
To determine whether the course of disease as assessed
by footpad swelling was correlated with the parasitic burden, L. major were quantitated from draining LN at various
time points after infection (Fig. 2). Mice vaccinated with
LACK DNA had an ~2-log reduction in parasite burden at
6 wk after infection compared with mice vaccinated with
control DNA. Similarly, mice vaccinated with LACK protein plus rIL-12 had a reduction in parasitic load compared
with those mice vaccinated with LACK protein alone. Finally, it should be noted that mice vaccinated with LACK
DNA or LACK protein plus rIL-12 continued to show
control of parasite replication for up to 10 wk after infection. Similar results were seen in a separate experiment
when parasitic burden was quantitated from the footpad lesions 8 wk after infection. Mice vaccinated with control
DNA had 2.1 × 109 viable parasites/mg of tissue. In contrast, mice vaccinated with LACK DNA were found to
have 4-log decrease (3.02 × 105 parasites/mg of tissue) in
parasitic burden from their footpad lesions.
To determine the optimal
amount of LACK DNA needed to confer protection, mice
were injected with either 10, 30, or 100 µg of LACK
DNA. To control for potential nonspecific immunostimulatory effects of bacterial DNA, control DNA was added to
some groups so that all mice received a total of 100 µg of
DNA. As shown in Fig. 3 A, LACK DNA vaccination induced effective immunity in a dose-responsive manner.
Thus, based on these experiments, we conclude that at least
100 µg of LACK DNA is required for a protective response.
As alluded to in the Introduction, IL-4 and IL-12 have been shown to be critical cytokines in controlling disease outcome to L. major infection. To evaluate whether combining cytokine DNA to
LACK DNA affected the immunity to L. major infection,
IL-4 DNA and IL-12 DNA were injected concomitantly
with LACK DNA. It should be noted that before these experiments, IL-4 and IL-12 DNA were shown to produce
biologically active protein from supernatants of COS cells
transfected with these respective DNAs (data not shown).
As shown in Fig. 3 B, footpad swelling in mice vaccinated with LACK DNA plus IL-12 DNA was not substantially
different than that seen in mice vaccinated with LACK
DNA plus control DNA. Because bacterial DNA (by virtue of their immunostimulatory sequences) has been shown
to be a potent inducer of IL-12 and IFN- It was of additional interest to determine whether effective immunity induced by LACK DNA
vaccination was both systemic and durable over a prolonged period after infection. Moreover, it was important
to determine whether the protective response induced by
LACK DNA vaccination was maintained when mice were
infected beyond 2 wk after the last vaccination. To this
end, mice were infected 5 wk after the last vaccination
with LACK DNA and measurements of footpad lesions
were continued for at least 20 wk after infection. As shown
in Fig. 4 A, mice vaccinated with LACK DNA maintained an effective immune response that was sustained for at least
20 wk, even when the infectious challenge was given 5 wk
after the last vaccination. The ability of LACK DNA to induce effective immunity at a site distant from the point of
vaccination was shown in a separate experiment by infecting mice in the opposite footpad to which they received
LACK DNA vaccination. As shown in Fig. 4 B, mice were
protected in a similar manner to those mice infected in the
same footpad.
Production of IFN-
Because cytokines
such as IL-4 and IFN-
It is clear from previous studies that the
cytokine milieu at the initiation of infection is critical in determining disease outcome (33). In this regard, antigen-specific in vitro production of IL-4 and IFN-
To assess the mechanism by which LACK DNA vaccination enhanced production of IFN-
In this report, we show that immunization of genetically
susceptible BALB/c mice using LACK DNA protected
virtually all of the mice against progressive, nonhealing infections with L. major. Furthermore, we report here an effective vaccine (given subcutaneously) in BALB/c mice
against cutaneous leishmaniasis where immunomodulating adjuvants such as IL-12 were not required. In the only reported use of DNA vaccination in this model, it was not
demonstrated that control of infection was effectively
achieved in a sustained manner (41). However, the immunity conferred by LACK DNA was maintained for at least
20 wk after infection and provided effective systemic immunity at a site distant from the point of vaccination. Furthermore, previous studies using LACK protein or SLA
combined with rIL-12 (28, 29) showed protective efficacy
when animals were infected 2 wk after the last vaccination.
In our experiments, both LACK protein plus rIL-12 and
LACK DNA induced a protective response when mice
were infected 2 wk after the last vaccination. Further studies are in progress to compare the ability of protein vaccination plus rIL-12 versus DNA vaccination to induce long lasting immunity. Finally, these data show that LACK
DNA immunization without adjuvants elicits a potent immune response that is similar to that induced by LACK
protein plus rIL-12, but is qualitatively very different from
that induced by LACK protein alone because the latter, as
previously reported and confirmed here, induced no protection whatsoever (29). Thus, these results clearly indicate that DNA vaccination can substitute for potential cytokine
adjuvants like IL-12.
DNA vaccination has been shown to elicit a long-lasting
immune response in a variety of animal models (42, 43).
Although the immunological mechanisms of DNA vaccination have still not been completely elucidated, there is
evidence to suggest that the route (18, 19) or the dose of
DNA play critical roles in determining the type of response
induced. In our initial studies, we were unable to show any
protective immunity when LACK DNA (5 µg) was administered using a gene gun (data not shown). These results
are consistent with previous work showing that this route (and/or dose) of immunization induces a preferential Th2-type response, which would fail to provide protective immunity in this model. By contrast, immunizing mice subcutaneously with LACK DNA allowed us to administer much
larger amounts of DNA, showing that protective immunity
was dose dependent. At least 100 µg of LACK DNA was
required for optimal protection. Based on the recent demonstration that bacterial DNA containing CpG motifs are
potent activators of the immune system (44), it is likely that
these immunostimulatory sequences (17) played a critical
role in the efficacy of the vaccine through their preferential
induction of IL-12 and IFN- Previous studies have shown that the cytokine milieu
present at the initiation of infection plays an important role
in the subsequent development of either a protective Th1
or a noncurative Th2 response (34). In particular, treatment of susceptible BALB/c mice with repeated doses of
IL-12 at the time of infection mediates a protective response and is associated with the production of Th1 type
cytokines (24, 25), whereas neutralization of IL-12 leads to
disease susceptibility in resistant mice (25). Alternatively, treatment of susceptible mice with anti-IL-4 at the time of
infection has been previously shown to enhance IFN- Our analysis of the immune response in protected mice
is consistent with the ability of LACK DNA (when given
by the appropriate route and amount) to induce a potent
Th1-type response. There was preferential induction of
LACK-specific production of IFN- Remarkably, the data also show that antigen-specific IL-4
production from lymph cells stimulated in vitro 6 wk after
infection was similar in all the groups. These observations
were confirmed in vivo by studying antibody isotypes. Although protected mice had an increase in IgG2a (consistent
with increased IFN- One final observation relates to the role of CD8+ T cells
in the immunity observed. Depletion of CD8+ T cells in
LACK DNA vaccinated mice at the time of infection abrogated immunity. In this regard, while previous work has
provided overwhelming evidence that CD4+ T cells play a
critical role in determining disease outcome in this model
(20, 53, 54), there is also evidence that CD8+ T cells
can mediate an anti-leishmanial effect in secondary responses (36). Moreover, although it is not yet clear how CD8+
T cells are mediating their effects in vivo, it has been
shown that parasite-specific CD8+ T cells can contribute to
the IFN- production results
in progressive infection and a fatal outcome (20). In addition, it has been demonstrated that manipulation of the
immune response at the time of infection with exogenous
IL-12 (24, 25), or inhibition of endogenous IL-4 (26, 27),
leads to protective immunity mediated by preferential induction of a Th1-type response. Furthermore, vaccination
with either soluble leishmanial antigens (SLA) or the single
parasite LACK protein in the presence of recombinant IL-12 protein has been shown to induce a protective Th1 response (28, 29).
, in response to antigenic stimulation (30). Moreover, BALB/c mice made tolerant to LACK by transgenic expression of LACK in the
thymus were found to be resistant to parasite infection,
suggesting that the early activation of LACK-reactive T
cells contributes to the initial cytokine milieu favoring a
nonhealing Th2-type phenotype (30). These observations were further extended in a recent report showing that early
IL-4 messenger RNA (mRNA) expression in response to
LACK was diminished in V
4-deficient BALB/c mice
(31). Taken together, these results suggest that early production of LACK-specific IL-4 from V
8+ V
4+ CD4+ T
cells in BALB/c mice is required for Th2 development and
susceptibility to infection. Thus, because altering the LACK-specific Th2 response in BALB/c mice induces resistance
to infection (29), we assessed the ability of LACK DNA
as a vaccine to induce protective immunity in BALB/c
mice infected with L. major.
173) promastigotes were grown at 26°C in 199 medium supplemented with
20% HI-FCS (Hyclone Laboratories, Inc., Logan, UT), 100 U/ml
penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM
Hepes, 0.1 mM adenine (in 50 mM Hepes), 5 µg/ml hemin (in
50% triethanolamine), and 1 µg/ml 6-biotin (M199/S). Infective-stage promastigotes (metacyclics) of L. major were isolated from stationary cultures (5-6 d old) by their lack of agglutination with 100 µg/ml of peanut agglutinin (Vector Laboratories, Inc., Burlingame, CA). Mice were challenged with 105 metacyclic
promastigotes in their hind footpads 2 wk after the boost. Weekly
footpad swelling measurements were recorded using a caliper.
20°C.
and
IL-4 were assessed by specific ELISA as previously described (32).
The lower limit of detection of IFN-
and IL-4 was 30 pg/ml
and 1.5 pg/ml, respectively.
LACK DNA Vaccination Induces Similar Protective Efficacy
Compared with LACK Protein Plus rIL-12.
Fig. 1.
LACK DNA vaccination provides similar protection as LACK
protein plus rIL-12 to BALB/c mice infected with L. major. BALB/c mice (n = 12/group) were initally immunized and boosted 2 wk later with LACK DNA, control DNA (100 µg), or with LACK protein (50 µg) with or without rIL-12 (1 µg). 2 wk after the boost, mice were challenged in the hind footpad with 105 live L. major metacyclic promastigotes. Weekly footpad measurements represent the average foot pad
scores ± SEM.
[View Larger Version of this Image (27K GIF file)]
Fig. 2.
Quantitation of viable parasite from draining lymph nodes.
Single cell suspensions were made from draining lymph nodes harvested from infected mice at various time points after infection. The number of
viable parasites was determined from the highest dilution at which promastigotes could be grown out 7 d of incubation. Data shown represents
an average of three mice ± one SD at each time point.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Cytokine DNA and
the amount of DNA used for
vaccination influence disease
outcome. (A) Mice (n = 12/
group) were immunized and
boosted 2 wk later with varying
doses of LACK DNA (10, 30, and 100 µg) in combination
with varying amounts of control
DNA to provide a total of 100 µg of DNA per vaccination. (B)
In the same experiment, mice (n
= 12/group) were immunized
and boosted 2 wk later with
LACK DNA (100 µg) plus 100 µg of control DNA, IL-4 DNA,
or IL-12 DNA. 2 wk after the
boost, mice were challenged in
the hind footpad with 105 live L. major metacyclic promastigotes.
[View Larger Versions of these Images (18 + 19K GIF file)]
, it is likely that
the effects of the additional control DNA is comparable to
the effect achieved by IL-12 DNA. Remarkably, the addition of IL-4 DNA significantly abrogated the protective efficacy of LACK DNA. This result is consistent with previous observations showing the potent effects of IL-4 in
regulating protective immunity to L. major. Moreover,
these data suggest that when sufficient amounts of IL-4 are
present, they exert a dominant effect over that of IL-12
(32).
Fig. 4.
LACK DNA vaccination induces a systemic and durable immune response to mice infected with L. major. (A) BALB/c mice (n = 6/ group) were initially immunized with LACK or control DNA (100 µg)
and boosted 2 wk later. 5 wk after the boost, mice were challenged in the
hind footpad with 105 live L. major metacyclic promastigotes. (B) In a separate experiment, BALB/c mice (n = 6/group) were initially immunized
and boosted 2 wk later with LACK or control DNA (100 µg) in the right
hind footpad. 2 wk after the boost, mice were challenged with 105 live L. major metacyclic promastigotes in the same or the opposite footpad to
which they recieved LACK DNA vaccination.
[View Larger Versions of these Images (17 + 22K GIF file)]
Production In
Vitro from Draining Lymph Node Cells of Mice Infected with L. Major.
and IL-4 from draining LN
was assessed after in vitro stimulation with LACK protein 6 wk after infection. As shown in Fig. 5 A, LN cells from
mice that developed a healing phenotype (i.e., LACK
DNA, LACK DNA plus IL-12 DNA, and LACK protein plus rIL-12 protein) made significantly more LACK-specific IFN-
compared with mice (control DNA or LACK
protein) with progressive disease (P < 0.005). Furthermore, mice vaccinated with LACK DNA plus IL-4 DNA
had a decrease in production of LACK-specific IFN-
, consistent with the fact that these mice were unable to
control infection (see Fig. 3 B). In the same experiments,
IL-4 production was also assessed from LN cells stimulated
with LACK antigen. Unexpectedly, production of IL-4
was similar in all groups regardless of the disease outcome
(Fig. 5 B); however, it should be noted that there was an
increase in the production of antigen-specific IL-4 from
mice vaccinated with LACK DNA plus IL-4 DNA. Finally, similar results with regard to production of IFN-
and IL-4 were observed using a class II MHC peptide for
LACK or SLA for stimulation, although the magnitude of
the SLA response was less than that seen in response to
LACK protein or specific peptide (data not shown).
Fig. 5.
In vitro production of IL-4 and IFN- from lymph node cells of vaccinated mice infected with L. major at 6 wk after infection. Individual
mice (n = 3) were euthanized and the draining lymph nodes were harvested 6 wk after infection. Single cell preparations were plated in triplicate in 96-well microtiter plates at 3 × 105 cells/200 µl in media alone or with LACK protein (10 µg/ml). 48 h later, supernatants were harvested and IFN-
and
IL-4 content were assayed by ELISA. Production of IFN-
in media alone was <30 pg/ml. Production of IL-4 in media alone was usually 125 pg/ml or
less. Data as shown represents the amount of IL-4 and IFN-
averaged from three individual mice ± SEM. *P <0.005 in comparing IFN-
produced
from mice vaccinated with LACK DNA or LACK protein plus rIL-12 versus that produced from mice vaccinated with control DNA or LACK protein
alone.
[View Larger Versions of these Images (38 + 35K GIF file)]
direct immunoglobulin class switching for IgG1 and IgG2a, respectively, we measured LACK-specific production of these antibody isotypes to provide an
indirect, but physiologic in vivo assessment of the pattern of cytokine production. As shown in Fig. 6 A, mice that
developed an effective immune response (LACK DNA
plus or minus IL-12 DNA or LACK protein plus rIL-12)
had substantially higher levels of LACK-specific IgG2a antibody titers compared with nonhealing mice (control
DNA or LACK protein). Furthermore, as shown in Fig. 6 B, it was remarkable that LACK-specific IgG1 remained elevated in mice that had developed a protective immune response consistent with the data shown above (see Fig. 5 B).
Finally, total serum IgE were found to be similar in all
groups of mice regardless of disease outcome, providing
further evidence that IL-4 levels in vivo were not substantially different in any of the mice (data not shown). These
results are consistent with the in vitro assessment of cytokine production shown above in that IFN-
is increased from groups that are protected compared with control
mice, but IL-4 is not significantly different among the various groups.
Fig. 6.
LACK-specific production of IgG1 and IgG2A in vaccinated
mice infected with L. major. Pooled sera (n = 7-12 mice per group) was
collected 6 wk after infection from mice vaccinated with LACK DNA
with or without IL-12 DNA, or LACK protein in the presence or absence of rIL-12. Sera was tested for LACK-specific antibody (A) IgG2a or
(B) IgG1. Data shown is an average of duplicate absorbance (OD) values
using serial fivefold dilutions.
[View Larger Versions of these Images (21 + 25K GIF file)]
Before Infection Is Predictive of
Disease Outcome.
were assayed
from draining LN of immunized mice before infection
with L. major. As shown in Fig. 7, mice that controlled infection (LACK DNA plus or minus IL-12 DNA or LACK protein plus rIL-12) made substantial amounts of IFN-
in
response to LACK protein, whereas nonhealing mice had
no detectable IFN-
. As a control, there was no production of IFN-
in any of the groups in response to SLA (data
not shown). These results suggest that enhanced production of LACK-specific IFN-
before infection is predictive
of disease outcome and further underscores the ability of
LACK DNA vaccination to induce IFN-
preferentially.
Fig. 7.
Enhanced production of IFN- before infection is predictive
of disease outcome. Pooled draining lymph nodes from mice (n = 5)
were harvested 2 wk after the boost and before mice were infected with
L. major. IL-4 and IFN-
protein production were determined in a similar manner to that outlined in Fig. 5.
[View Larger Version of this Image (27K GIF file)]
, mice were vaccinated with LACK DNA
and treated weekly from the time of initial vaccination until 4 wk after infection with a neutralizing antibody against
IL-12. As shown in Fig. 8 A, anti-IL-12 treatment completely abrogated protection. Furthermore, vaccinated mice
treated with anti-IL-12 had a striking inhibition of in vitro
production of IFN-
(Fig. 8 B), suggesting that LACK
DNA induced protective immunity through IL-12-dependent production of IFN-
. In addition to the ability of
DNA vaccination to enhance Th1 responses, it has also
been shown to be a potent inducer of MHC class I-restricted
CD8 responses (13). Furthermore, because CD8+ T cells
typically secrete IFN-
and have been previously shown to
have a role in secondary infection to L. major (36), we
evaluated the effect of CD8+ T cell depletion at both the
time of vaccination and the time of infection. Depletion of
CD8+ T cells at the time of vaccination would potentially
eliminate an initial source of IFN-
that may be important
in generating a strong Th1 response. Alternatively, by delaying the depletion of CD8+ T cells until the time of infection, we could evaluate the requirement for these cells in the
effector phase of the response while allowing for the potential production of IFN-
from CD8+ T cells up to the time
of infection. Surprisingly, anti-CD8 treatment at the time
of infection completely abrogated the protective response elicited by immunization with LACK DNA (Fig. 9). These
results suggest that CD8+ T cells have a role in mediating
an effective immune response during a primary infection
from mice vaccinated with LACK DNA.
Fig. 8.
LACK DNA vaccination confers protection in an IL-12-dependent manner. (A) BALB/c mice immunized and boosted 2 wk later with LACK or control DNA (100 µg) were challenged with live 105 L. major
metacyclic promastigotes 2 wk after the boost. Some of the vaccinated
mice (n = 10/group) were treated with 1 mg of anti-IL-12 intraperitoneally weekly from the time of vaccination to 4 wk after infection. (B) In
the same experiment, draining lymph nodes from individual mice were
harvested 6 wk after infection and production of IL-4 and IFN- were
determined in a similar manner to that outlined in Fig. 5. *P <0.005 in
comparing IFN-
produced from mice vaccinated with LACK DNA
versus that produced from mice vaccinated with LACK DNA plus anti-
IL-12.
[View Larger Versions of these Images (25 + 26K GIF file)]
Fig. 9.
CD8+ T cells have an important role in mediating protection
induced by LACK DNA vaccination. Mice (n = 10/group) receiving LACK or control DNA vaccination were treated with anti-CD8 antibody intraperitoneally starting at the time of vaccination (V) or at the time of
infection (I) and then weekly until 4 wk after infection.
[View Larger Version of this Image (27K GIF file)]
(45). Thus, the critical
protective dose of subcutaneously administered LACK
DNA was due to either a requirement for a threshold dose of immunostimulatory sequence, antigen encoding sequence,
or both.
leading to control of infection (26, 27). In addition, anti-
IL-4 treatment at the time of infection was recently shown
to be important in maintaining early responsiveness to IL-12 (35). Thus, successful vaccination against this infection
must provide sufficient IL-12 to overcome the potent immunoregulatory effects of the early IL-4 burst that accompanies inoculation of these parasites into BALB/c mice.
before infection (Fig.
7), and this response was sustained at least 6 wk after infection. The role of IL-12 in mediating both the IFN-
response and the protective effect induced by LACK DNA vaccination was demonstrated by the fact that neutralizing
IL-12 in vivo decreased the LACK-specific IFN-
production by LN cells, and completely abrogated protection.
), they also had similar levels of IgG1
and IgE (data not shown), as the unprotected mice, suggesting that IL-4 was present as well. The fact that IgG1
was increased in healing mice in which IFN-
was enhanced is consistent with previous work showing that IL-12 treatment could induce an increase in both IgG1 and
IgG2a (49). In addition, an assessment of cytokine mRNA
from draining LN cells showed similar levels of IL-4
mRNA as assessed by semiquantitative PCR, further substantiating that IL-4 was similar in all the groups (data not shown). It is possible that the significant levels of IL-4 detected 6 wk after infection in the protected groups was because LACK DNA vaccination was unable to influence all
LACK-reactive T cells that have been recently shown to be
highly enriched in their capacity to produce IL-4 (30, 31).
Taken together, these data suggest that in our DNA vaccine model, the relative ratios of IL-4 and IFN-
predict
disease outcome and the ability to control progressive infection may not be dependent on the development of a
true polarized response with the complete absence of IL-4,
consistent with previous studies (50). In this regard, it is
important to note that despite control of footpad swelling
and a reduction in parasite growth in both the footpad and
draining node, there was still a significant number of viable
parasites present in these sites, even 10 wk after infection.
Additional studies are in progress to determine whether
neutralization of IL-4 at the time of LACK DNA vaccination leads to more efficient reduction in parasitic burden
with more complete polarization of the immune response.
response in a secondary infection, suggesting
this as a possible mechanism (38). In the present study, the
fact that mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of infection were no longer
protected provides evidence that CD8+ T cells can also
mediate protective immunity in a primary infection. Because DNA vaccination is a potent inducer of IFN-
production as well as MHC class I- and class II-restricted T
cell responses, we cannot be certain as to the relative contribution of each of these mechanisms in mediating the
protective response. However, as noted above, because
neutralizing IL-12 in vivo substantially diminished IFN-
production and completely abrogated protection conferred
by LACK DNA, it is likely that contribution of CD8+ T
cells in mediating protection is through an IL-12-dependent enhancement of IFN-
production, although an IL-12-dependent cytolytic mechanism cannot be excluded at
this time. In ongoing experiments, in assessing in vitro production of IFN-
from LN cells of LACK DNA vaccinated mice after infection, it was noted that addition of either anti-CD4 or anti-CD8 antibodies to the cultures caused a significant decrease in IFN-
production (data not shown).
Based on these data, we would speculate that a critical
threshold of IFN-
is required for control of infection and
that DNA vaccination elicits IFN-
from both CD4+ T
cells and CD8+ T cells in an IL-12-dependent manner. By
contrast, we would speculate that protective immunity
achieved by treating mice with LACK protein plus IL-12
protein is likely to result from IFN-
produced primarily by CD4+ T cells. Thus, it is possible that vaccination with
DNA in BALB/c mice may induce protection to L. major
by a somewhat different mechanism than vaccination with
protein plus adjuvant. Nevertheless, these data indicate that
DNA vaccination can substitute for conventional protein
vaccination requiring a cytokine adjuvant in providing effective immunity against an intracellular parasite.
Address correspondence to R.A. Seder, NIAID, NIH, Building 10, Room 11C215, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-402-4816; FAX: 301-496-7383; E-mail: rseder{at}nih.gov
Received for publication 23 June 1997 and in revised form 14 July 1997.
S.L. Reiner is supported by the Burroughs Wellcome Fund and the National Institutes of Health (AI-01309). N. Glaichenhaus is supported by grants from the Conseil Régional de la Région PACA (N. Glaichenhaus) and from the Ministère de l'Education Nationale, de la Recherche et de l'Enseignement Supérieur (N. Glaichenhaus).We thank Dr. Ethan Shevach and Dr. Richard Locksley for helpful discussions.
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