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*
Division of Immune Regulation and
Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and
Department of Pathology, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting lymphocytes and enhanced NK cell activity
in the draining popliteal node. In contrast, resistant B10.D2 mice
given i.m. injections with a recombinant adenovirus-expressing IL-4
displayed greater susceptibility to disease, and severe lesions were
produced in some of the infected animals. These results suggest the
potential use of recombinant adenoviruses expressing cytokines as
potent immunomodulatory agents for the generation of protective immune
responses against intracellular pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production being detected. In contrast, parasitic infection in
other strains of mice, such as B10.D2, C3H, C57BL/6, C57BL/10, and 129,
produces self-healing lesions associated with a strong Th1
cell-mediated immune response with production of high levels of IFN-
and other inflammatory cytokines (2). In vivo and in vitro studies
concerning the initiation events that cause Th1 or Th2 cell development
in L. major-infected mice have demonstrated that IL-4 is the
main cytokine inducer of a Th2 response, while IL-12 and IFN-
promote Th1 cell expansion (3). In fact, therapies directing immune
response toward type 1 immunity, including the use of rIL-12 protein as
a vaccine adjuvant, have been used successfully to protect BALB/c mice
against disease (4, 5, 6). However, IL-12 therapies generally require
multiple injections, owing to the short half-life of recombinant
proteins in vivo. A therapeutic regimen based on a single injection of
IL-12 in a suitable vehicle would be a major advance in the prevention
of leishmaniasis. Adenoviruses-expressing cytokines are useful agents
for delivery of cytokines due to their higher levels of gene expression
and their localization at the site of inoculation (7). Using the Ad5IL-123 vector (a recombinant adenovirus type 5-expressing IL-12), we show that a single ipsilateral-localized i.m. injection of the recombinant vector administered on the same day as L. major parasitic challenge prevented the development of lesions in the footpads of BALB/c mice and the spread of parasitic infection to peripheral organs. The Ad5IL-12 vector treatment induced immune deviation that elicited a protective Th1 immune response against the L. major parasite in the normally Th2-responsive BALB/c strain mice.
Interestingly, in parallel experiments designed to test the effects of vector-derived IL-4 expression, B10.D2 mice (a MHC-matched disease-resistant strain) were rendered susceptible to L. major infection after receiving a single i.m. injection of the Ad5IL-4 vector with parasite challenge. This is the first report demonstrating that overexpression of IL-4 can increase disease susceptibility in a resistant mouse strain. Taken together, these results suggest that recombinant adenovirus vectors expressing cytokines act as powerful immunomodulatory agents and can serve a useful role as "molecular adjuvants" to generate protective immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All recombinant viruses were propagated on 293 cells and purified by cesium chloride gradient centrifugation. Construction and characterization of the Ad5 vectors expressing IL-12 and IL-4 have previously been described (8, 9). In brief, the Ad5IL-12 vector contains an expression cassette for the p35 subunit of IL-12 in the E1 region, and the IL-12 p40 subunit in the E3 region. The Ad5IL-4 vector contains an expression cassette for IL-4 in the E1 region. The DL70–3 control virus is a Ad5 variant deleted in the E1 region (10). For treatment, mice were injected with 5 x 108 plaque-forming units (pfu) of recombinant adenovirus in 50 µl PBS into the femoral bicep muscle of the left hind leg on the same day as footpad injection of the L. major promastigotes.
Animals and parasites
BALB/c and B10.D2 mice (6–8 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the La Jolla Institute for Allergy and Immunology animal facility. Infections were performed with stationary-phase promastigotes of L. major (strain WHOM/IR/-/173) grown at 28°C in M199 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 40 mM HEPES, 0.1 mM adenine, and 5 µg/ml hemin. Animals were injected in the left hind footpad with 2 x 106 stationary-phase L. major promastigotes, and the course of the disease was monitored at weekly intervals by measurement of footpad thickness with a metric caliper. All work was performed in accordance with La Jolla Institute for Allergy and Immunology guidelines for animal use and care.
Parasite burden
Six weeks postinfection, parasite loads were determined by limiting dilution analysis to measure the number of viable L. major parasites in the tissues of infected mice as previously described (11). Briefly, draining lymph nodes and spleens were homogenized in M199 medium with supplements, and serial fivefold dilutions of the homogenates were plated in flat-bottom 96-well microtiter plates and kept at 28°C for 1 week. The wells were assessed for growth of L. major promastigotes microscopically at the highest dilution at which three of eight replicates were positive.
Collection and processing of tissues
At designated periods, blood samples were collected from the retroorbital plexus of mice treated with recombinant adenovirus; or, mice were sacrificed for the preparation of a muscle tissue extract. In addition, draining lymph nodes were collected for cytokine ELISA-spot assays. Frozen tissues were homogenized in PBS (3 ml per muscle) containing 100 µM PMSF and 10 µg/ml aprotinin. The homogenate was freeze-thawed three times and cleared by centrifugation. Aliquots were stored at -20°C until analyzed.
ELISA
IL-12 and IL-4 levels in muscle homogenates and serum were measured by a sandwich ELISA. Briefly, Nunc-Immuno Plate MaxiSorp F96 (Nunc, Roskilde, Denmark) were coated with anti-IL-12 (p35/p70; clone Red-T/G297-289; PharMingen, San Diego, CA) or anti-IL-4 Abs (purified from 11B11 cell supernatants). After blocking with PBS containing 10% FBS, muscle homogenates or sera were added O/N at 4°C. Plates were extensively washed with PBS-Tween and incubated with biotin-conjugated anti-IL-12 (p40/p70; clone C17.8; (PharMingen) or anti-IL-4 (PharMingen) Ab. Finally, the plates were washed and developed using avidin-peroxidase and 2-2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate (Sigma, St. Louis, MO). OD was measured at 405 nm, and the values were determined against a recombinant protein standard.
Cellular ELISA-spot assay
IFN-- and IL-4-producing cells were enumerated in recombinant
adenovirus-treated mice by a cellular ELISA-spot assay as described
(12). Briefly, lymph node cells (5 x 106/ml) were
cultured for 48 h in 24-well plates with media alone,
paraformaldehyde-fixed L. major parasites, or
Leishmania-activated protein kinase C (LACK) peptide
160–173. Millititer HA Nitrocellulose plates (Millipore, Bedford, MA)
were coated O/N at 4°C with anti-IFN- (purified from R46A2
supernatants) or anti-IL-4 Ab. Plates were blocked, and
Ag-stimulated cells were added at different concentrations for 24
h at 37°C. The wells were then incubated with biotin-conjugated
anti-IFN- (purified from XMG1.2 supernatants) or anti-IL-4
Ab followed by incubation with avidin-peroxidase (Vector, Burlingame,
CA). Spots were developed by the addition of 400 µg/ml of
3-amino-9-ethylcarbazole substrate (Sigma) and enumerated by a
computerized image analysis system (Lightools Research, Encinitas, CA)
using the image analyzer program NIH Image 1.61 (National
Institutes of Health, Bethesda, MD).
Determination of NK cell activity
NK cytotoxic activity was determined as previously described (4). 51Cr-labeled YAC-1 cells were used as targets in a standard 4-h 51Cr release assay at several E:T ratios for cells isolated from the popliteal lymph node of recombinant adenovirus-treated and control mice. Briefly, 1 x 107 target cells were labeled for 2 h with 500 µCi of sodium chromate in 0.5 ml of RPMI 1640 media (HyClone, Logan, UT) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME. Samples were recorded in triplicate at each E:T ratio using 5 x 103 YAC-1 target cells per well in V-bottom microtiter plates. The percentage of specific 51Cr release was calculated using the following formula: 100 x [(cpm test sample - cpm medium control)/(cpm 1 N HCl sample - cpm medium control)].
FACS analysis
To characterize the lymphocytes recovered from popliteal nodes
in 6-wk recombinant adenovirus vector-post-treated animals, cells were
analyzed against a panel of mAbs (PharMingen) specific for CD3, CD4,
CD8, CD45RB, Ly49C, DX5, B220, and TCR Vß8.2, Vß4, and V
8 chains
using a FACScan flow cytometer (Becton Dickinson, Mountain View,
CA).
Statistical analysis
Data are expressed as the mean ± SEM for each group. Statistical analysis was performed using Statview 4.5 programs from Abacus Concepts (Berkeley, CA). A Student t test was used for the final determination of significance testing the effects of Ad5IL-12 and Ad5IL-4 treatments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because IL-12 had been shown to be an important cytokine in
directing Th1 immune responses that are associated with protection
against leishmaniasis, we performed experiments to explore the role of
the Ad5IL-12 vector vs controls as a way to prevent parasitic lesion
development. For this study, BALB/c mice were infected with 2 x
106 L. major promastigotes into the left hind
footpad and injected i.m. with a 5 x 108 pfu dose of
recombinant adenovirus on the ipsilateral side. As shown in Fig. 1
A, BALB/c mice receiving a
single Ad5IL-12 vector i.m. injection were able to effectively control
the development of parasitic lesions. In these experiments, 17 of 18
animals were protected and did not develop footpad lesions, whereas all
control vector (DL70-3-injected, vector without cytokine insert) and
parasite-infected mice developed significant lesions by 6 wk leading to
termination of the experiment. The protection afforded by the Ad5IL-12
vector was durable in treated mice because infection remained under
control for as long as 1 year (in 12 mice followed). Of these 12 mice,
two groups of 4 mice each were rechallenged with an additional
parasitic dose of 2 x 106 L. major at
either 6 wk or 4 mo after the initial Ad5IL-12 vector treatment. All of
these animals were protected against infection after the secondary
challenge, indicating the development of immunologic memory to the
pathogen (data not shown). To be effective, the IL-12 vector had to be
delivered close to the time of L. major challenge. By day 3
post-Ad5IL-12 vector administration, very little protection was
provided against leishmaniasis, and if L. major infection
was performed 1 wk after Ad5IL12 i.m. injection, protection was absent
(Fig. 1B). Simultaneous i.m. administration of Ad5IL-12
vector contralateral to the L. major-infected footpad did
not afford any significant protection against disease (data not shown).
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Because IL-12 expression derived from i.m. injection of the
Ad5IL-12 vector blocked the progression of leishmaniasis in susceptible
BALB/c mice, we attempted to test if IL-4 vector-derived expression
could induce susceptibility in the normally resistant MHC-matched
B10.D2 mice. It has been shown that systemic administration of blocking
Abs against Th1-type cytokines could divert immune response to the
parasite and allow disease to progress in resistant strains (13).
However, to date, attempts at inducing disease with rIL-4 protein has
failed in resistant mouse strains (14, 15). Here, we report that a
single i.m. injection of the Ad5IL-4 vector to resistant B10.D2 mice
caused 3 of 11 mice to develop open necrotic lesions (Fig. 2A), with the
remaining 8 animals all showing significant increases in footpad
swelling compared with the vector-only controls. Fig. 2B
shows comparative lesions between "protected" BALB/c and
"susceptible" B10.D2 mice after Ad5IL-12 or Ad5IL-4 treatment,
respectively, vs DL70–3 vector control. One of the most severely
afflicted Ad5IL-4-treated animals had a lesion of 7.3 mm in diameter
and was sacrificed at 6 wk. Limiting dilution analysis was performed to
determine residual parasite burden on cells isolated from the
ipsilateral draining popliteal nodes of the animals at this time point
(Fig. 2C). The Ad5IL-4 vector-treated animals had 600-fold
(
2.5 log) higher parasite titers in their draining nodes. For one
experiment, disease progression was monitored until 15 wk
postinfection, when lesions had completely receded in all animals
except in one of the Ad5IL-4-treated mice. Quantification of parasite
levels in draining lymph nodes at 15 wk postinfection also demonstrated
a 125-fold increase in the Ad5IL-4 vector-treated group compared with
other groups. Of interest, the most severely afflicted animals from the
Ad5IL-4-treated group were found to have L. major
promastigotes detectable in the spleen, which were not present in other
animals from control groups (data not shown). These data indicate that
IL-4 expression not only exacerbates disease locally but renders
animals more susceptible to parasitic spread to peripheral organs.
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In contrast to the enhancing effects of IL-4 expression on
parasitic burden in B10.D2 mice, Ad5IL-12 treatment reduced parasitic
infection in the BALB/c strain. Mice infected with L. major
and receiving additional treatment with Ad5IL-12, adenovirus vectors
DL70–3, Ad5IL-4, or no adenovirus treatment were assayed for parasitic
burden in lymph node and spleen at 6 wk postinfection (Fig. 3, A and B). At
this time point, all control mice had pronounced footpad lesions, with
some animals becoming moribund and requiring euthanasia. Meanwhile, all
of the experimental Ad5IL-12-treated mice in this experiment were
lesion-free and showed no signs of significant morbidity. Parasitic
titers in the draining popliteal lymph node of Ad5IL-12-treated mice
(Fig. 3A) were dramatically lower than those of
control-treated mice (by 3 log = 3125-fold). The titers
detected in the Ad5IL-12-protected BALB/c mice were similar to those
that are recovered in healing B10.D2 animals after infection. Gross
morphologic analysis of vector-treated mice revealed splenomegaly in
controls vs Ad5IL-12-treated BALB/c mice. Limiting dilution analysis
revealed that untreated animals with splenomegaly had a 125-fold
increased titer of parasites in the spleen compared with
Ad5IL-12-treated mice, in which seven of eight animals were parasite
free (Fig. 3B). These results indicate that vector-derived
IL-12 expression had a significant negative impact on the growth and
spread of L. major parasites during the course of infection
in susceptible BALB/c mice.
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The cytokine expression patterns of IL-12 in BALB/c and IL-4 in
B10.D2 mice were determined following vector administration. For time
course studies, expression was followed over a 1-wk period in sera or
tissue extracts prepared from recombinant adenovirus-treated animals
(Fig. 4). At the 1-day point, IL-12
expression reached 24.9 ± 3.6 ng levels within the muscle tissue
in BALB/c mice receiving a 5 x 108 pfu dose of the
Ad5IL-12 vector (Fig. 4A). IL-12 muscle expression peaked at
day 1 followed by a dramatic decline on day 3. This diminished IL-12
expression at the day-3 time point correlates well with the timeframe
when reduced protection against parasite infection occurs (Fig. 1
B).
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The peak level of IL-4 expression in B10.D2 mice, given an i.m.
injection of the Ad5IL-4 vector, also occurred at day 1, reaching 2.9
± 1.1 ng per injected muscle tissue and returning to baseline levels
by day 3 (Fig. 4B). As seen for cytokine expression after
Ad5IL-12 treatment, the serum levels of IL-4 in B10.D2 mice were
significantly lower and of shorter duration than levels achieved at the
injected site.
The efficacy of the Ad5IL-12 vector in the prevention of
leishmaniasis in BALB/c mice correlates with increasing numbers of
IFN--producing cells
Because dramatic differences in disease outcomes were evident
after i.m. injection with adenovirus vectors in BALB/c and B10.D2 mice,
an ELISA-spot cellular assay was used to study the frequency of
Ag-specific IFN-- and IL-4-secreting lymphocytes recovered from
draining lymph nodes postinfection. As shown in Table I, Ad5IL-12 vector treatment had a major
impact on the development of Ag-responsive lymphocytes. In both BALB/c
and B10.D2 mice, vector-derived IL-12 expression caused a major shift
in the background response to L. major and LACK peptide
160–173 toward a Th1 immune profile. The IFN-:IL-4 ratio in BALB/c
mice shifted from 0.18 to 4.42 in response to fixed L. major
parasites and from 0.47 to 16.70 for LACK peptide 160–173. Likewise,
the Th1 response in B10.D2 mice was also enhanced after IL-12 vector
treatment. IFN-:IL-4 ratios to fixed parasites increased from 1.71
to 16.33 and against LACK peptide 160–173 from 0.25 to 9.65. LACK has
been reported to be the target Ag in the induction of Th2 immune
responses in susceptible BALB/c mice (16). The Th1-induced deviation of
response to LACK peptide 160–173 may account for the changes seen in
the pathogenicity of the disease.
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:IL-4 production significantly from that of the
DL70-3 control. However, IL-4 expression had a profound influence over
disease progression, with a major change in the disease course
occurring at 3 wk (Fig. 2A). Cytokine stimulation of B cell
lymphopoiesis has been demonstrated to aggravate
leishmaniasis (17). We are currently investigating whether similar
effects are involved for IL-4 expression. Intramuscular injection of the Ad5IL-12 vector results in increased NK cell activation in the popliteal lymph node
It had been previously demonstrated that rIL-12 protein
administration either alone or in conjunction with soluble
Leishmania Ag could induce a NK cell response that has been
shown to be involved in the protection of BALB/c mice against L.
major infection (4). To determine whether i.m. injection with the
Ad5IL-12 vector could similarly influence the NK response, we measured
NK cell cytotoxic activity recovered in popliteal lymph node cells
using 51Cr-labeled YAC-1 cells as targets from mice that
were either naive, Ad5IL-12 vector-treated without parasitic infection,
L. major-infected also treated with IL-12 vector
control vector (DL70-3), or no adenovirus treatment (Fig. 5).
NK activity in Ad5IL-12-treated mice was
30-fold higher than control-treated mice as measured by the number
of effectors required for equivalent lysis of a fixed number of target
cells. The background NK response from uninfected BALB/c controls was
low and consistent with the activities previously reported for response
in this strain (18).
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Although no apparent change in lymph node cellular composition
occurred by 1 wk following infection with several phenotype markers
studied (data not shown), by 6 wk a clear difference was present. An
increase in the proportion of B220+ cells (a pan B cell
marker) was evident in L. major-infected compared with
Ad5IL-12-treated animals (Fig. 6). In
Ad5IL-12 treated mice, 15% of the lymph node cells stain for
B220 (normal phenotype) compared with 28.5% in the L.
major-infected BALB/c. T cells were the predominant population of
cells isolated from the popliteal nodes of Ad5IL-12-treated BALB/c
mice, accounting for 56% of the total staining population. In
comparison, a smaller proportion, 45% of the cells isolated from
L. major-infected animals, stained for CD3. No DX5-positive
cells were detected in the analyzed population.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Long-lasting immunity was conferred on susceptible BALB/c mice treated here with the Ad5IL-12 vector, as was previously demonstrated using DNA vaccination encoding the LACK gene (19). In the vaccination with LACK DNA, mice were reported disease-free for up to 20 wk. In our analysis, Ad5IL-12 vector-treated mice have remained healthy with no evident disease for >1 year. In addition, protected mice have an established immunity to subsequent infection. This efficient generation of a protective immune response by the Ad5IL-12 vector administered at the same time of L. major infection makes it an attractive candidate agent for use as an adjuvant in future vaccines.
Our data suggest that the adenovirus vector-derived IL-12 expression
mediates protection at both the innate and adaptive levels of immunity.
The early containment of parasites at draining lymph nodes and at the
local site of injection is a feature of resistant strains, while in
BALB/c mice L. major parasites can be recovered in the
spleen, lungs, and bone marrow 24 h after infection (20). It was
found that the containment of infection depended on the development of
a Th1 population in disease-susceptible BALB/c mice by rIL-12
administration. This cytokine causes T cells and NK cells to
proliferate (21) and secrete IFN- (22), which helps to create the
necessary milieu for Th1 skewing. In our hands, Ad5IL-12 treatment
similarly enhanced NK activity in addition to deviating the normal
BALB/c Th2 pattern of Ag-specific lymphocyte response toward Th1.
In previous reports describing the prevention of leishmaniasis in BALB/c mice, several administrations of rIL-12 were required before an effect was evident (6). IL-12 protection was possible only in a narrow window of time (between 16 and 48 h after infection), after which Th2 cells are already committed to a type 2 response and treatment is ineffective (16, 23). In our protocol, a single i.m. injection gave rise in 24 h to a local IL-12 level of cytokine that was enough to prevent disease. Of importance and relevance to potential therapy in humans, the adenovirus vector-induced IL-12 expression was found to be highly localized to the site of injection in the muscle. The fact that simultaneous i.m. administration of Ad5IL-12 vector contralateral to the L. major-infected site did not protect against disease indicates that colocalized expression of the cytokine with the Ag is an important parameter in the use of this vector to elicit protection against infection. This effect of targeted and localized transgene expression by recombinant adenovirus vectors is a highly desirable feature for expression of cytokines with potential toxicity. In other studies, systemic administration of rIL-12 had been found to be associated with the induction of anemia, leukopenia, thrombocytopenia, and splenomegaly among other symptoms (24, 25, 26). Recently, a phase I trial in cancer using rIL-12 had to be temporarily suspended owing to apparent toxicity (27). Unlike these earlier studies, the adenovirus vector-derived IL-12 expression induced no apparent toxicity in our experiments. In fact, the splenomegaly that occurs as a consequence of parasitic spread to peripheral organs during leishmaniasis in BALB/c mice was prevented by Ad5IL-12 treatment.
The role of IL-4 in inducing susceptibility to leishmaniasis has been a topic of much discussion, and its effect is still unclear. IL-4 has been shown to enhance Th2 immune responses, which are generally considered nonprotective against L. major parasites. However, a recent publication demonstrated that C57BL/6 mice (a L. major-resistant strain) had increased levels of IL-4 mRNA in the first week of infection but still controlled disease development (28). Systemic administration of IL-4 had no effect on disease induction in these mice (14). In another resistant strain (C3H/HeN), i.p. injection of rIL-4 was insufficient to generate disease susceptibility, although the normal Th1 response was curtailed (15).
However, a role for IL-4 in the induction of leishmaniasis has been indicated. Intralesional delivery of rIL-4 in BALB/c mice has been shown to exacerbate disease when administered at an early stage (29). Further, administration of anti-IL-4 Abs was shown to prevent lesion development in BALB/c mice. However, previous attempts to render resistant strains, such as B10.D2, susceptible to infection by IL-4 administration have been unsuccessful. Interestingly, administration of the Ad5IL-4 vector simultaneously to infection increased disease susceptibility in B10.D2 mice. This is the first report in which IL-4 overexpression has been described to influence leishmaniasis in resistant strains in such a dramatic fashion. It is possible that in previous studies rIL-4 administration failed to influence leishmaniasis in resistant mouse strains due to intrinsic problems with delivering and sustaining recombinant cytokine levels at the local tissue site. Further, it is possible that the temporal aspects of IL-4 administration may be important. It has recently been described that an early burst of IL-4 expression within 16 h of L. major infection can render BALB/c mice unresponsive to IL-12 (23). This effect may reflect IL-4-mediated down-regulation or modification of the IL-12R ß2-chain (30).
Phenotypic cellular analysis at 1 wk following treatment with Ad5IL-12 revealed no dramatic differences in TCR expansion or memory markers vs control BALB/c mice; however, an increase in B220+ cells was present at 6 wk in the L. major-infected control mice. This difference in B220 expression may reflect changes in the number of B cells. B220 has been described to be expressed on NK cells; however, we did not detect DX5-positive cells in the analyzed population. Previous work has indicated a deleterious effect associated with the presence of B cells in murine leishmaniasis, and a pronounced effect for B cells in efficiently activating IL-4 production by T cells in BALB/c mice (31). An important role for B cells in the pathogenesis of disease is in accord with the previous observation that depletion of B cells in BALB/c mice reduced lesion size (32). This effect was confirmed in a scid mouse model where cotransfer of B cells made resistant mice susceptible to L. major infection (33). It is possible that some of the protective effects attributed to Ad5IL-12 vector therapy involve its influence on B cell development during parasitic infection. One of the more intriguing possibilities is that both the IL-4 and IL-12 effects are due in part to either direct or indirect influence on B cell compartments and their activation.
Of relevance to future studies using adenovirus vectors to mediate cytokine delivery, differences in the IL-12 expression pattern between this study and our previous one using Ad5IL-12 as an intratumor treatment should be discussed. Intramuscular injection with the Ad5IL-12 vector produced lower levels of cytokine of shorter duration than those previously seen after intratumor injection (34). This may reflect a trivial difference in the detection Ab used (a p40 subunit-specific Ab was used in the previous study), but may be accounted for by differences in cell-specific susceptibility to recombinant adenovirus vector infection that may exist within tissues. One further possibility is that vector-derived cytokine expression was influenced by the tissue microenviroment. IL-10 is known to diminish antigenic display and tumor-mediated immune responses and could account for the higher levels of IL-12 expression detected after intratumor administration. Immune responses generated against adenovirus vectors have already been shown to influence transgene expression (35). The DL70-3 control vector treatment in both BALB/c and B10D2 mice induced some background immune responses. Small protective effects of the control DL70-3 vector were previously seen after direct tumor injection (34).
In conclusion, an increasing number of gene transfer vectors are currently being developed to deliver transgenes including cytokines as therapeutic agents. Of these, the development of short-term, focused special delivery systems in the form of adenovirus vectors holds much promise (36, 37, 38). Recombinant adenoviruses have already been used successfully as vaccines in a number of infectious disease models (38, 39, 40, 41) and recently in cancer gene therapy (8, 34, 42). In our initial report (7), the first describing adenovirus vectors expressing cytokines, we proposed that these vectors would be ideally suited for delivering cytokines as a transient and localized type of adjuvant therapy, particularly those using cytokine genes that may induce systemic immunotoxicities. With respect to leishmaniasis, a previous report demonstrated BALB/c mice infected with L. major were successfully treated with IL-12 and the antimonial drug, Pentostam (43). This also suggests that the adenovirus-mediated deliver of IL-12 might be a useful cotherapy. Adjuvant therapy, delivered by cytokine vectors, can provide an explosive level of agent, which in the case of a localized tumor, for example, in breast or prostate tissue, could establish a beneficial and rapid therapeutic milieu. These vectors can transduce both quiescent and replicating cells and results in the carriage of the adenovirus genome as episomal DNA, which makes this vector more suitable for delivering effector cytokines owing to its lack of integration. Finally, adenovirus vectors (inherent to DNA viruses) have properties selected by evolution for the delivery of transgenic material to nuclear compartments. In fact, adenovirus vector therapy is essentially a more efficient DNA vaccine. With the recent reported success of DNA vaccination encoding the LACK transgene (19), a direct comparison between DNA vaccines and recombinant adenovirus vectors to evaluate the efficacy of each approach would be of interest. Indeed, it may be advantageous to use naked DNA as a recall for recombinant adenovirus vector-induced responses, to avoid subsequent immunity to the vector. Regardless, our results support the development of an adenovirus vector vaccine incorporating cytokine and Ag that should be a highly efficient alternative to conventional vaccination in the prevention of leishmaniasis; this work is in progress.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Raja Gabaglia, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
3 Abbreviations used in this paper: Ad5IL-12, adenovirus type 5-expressing IL-12; Ad5IL-4, adenovirus type 5-expressing IL-4; pfu, plaque-forming units; O/N, overnight; LACK, Leishmania-activated protein kinase C.
Received for publication May 18, 1998. Accepted for publication October 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J. Immunol. 143:266.[Abstract]
8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
in vivo. Int. Immunol. 6:157.This article has been cited by other articles:
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  C. Raja Gabaglia, Y. Diaz de Durana, F. L. Graham, J. Gauldie, E. E. Sercarz, and T. A. Braciak Attenuation of the Glucocorticoid Response during Ad5IL-12 Adenovirus Vector Treatment Enhances Natural Killer Cell-Mediated Killing of MHC Class I-Negative LNCaP Prostate Tumors Cancer Res., March 1, 2007; 67(5): 2290 - 2297. [Abstract] [Full Text] [PDF]
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 Y. Diaz-de-Durana, G. T. Mantchev, R. J. Bram, and A. Franco TACI-BLyS signaling via B-cell-dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo Blood, January 15, 2006; 107(2): 594 - 601. [Abstract] [Full Text] [PDF]
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 T. A. Braciak, B. Pedersen, J. Chin, C. Hsiao, E. S. Ward, I. Maricic, A. Jahng, F. L. Graham, J. Gauldie, E. E. Sercarz, et al. Protection Against Experimental Autoimmune Encephalomyelitis Generated by a Recombinant Adenovirus Vector Expressing the V{beta}8.2 TCR Is Disrupted by Coadministration with Vectors Expressing Either IL-4 or -10 J. Immunol., January 15, 2003; 170(2): 765 - 774. [Abstract] [Full Text] [PDF]
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 E. F. Verdu, Y. Deng, P. Bercik, and S. M. Collins Modulatory effects of estrogen in two murine models of experimental colitis Am J Physiol Gastrointest Liver Physiol, July 1, 2002; 283(1): G27 - G36. [Abstract] [Full Text] [PDF]
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 H. W. Murray Clinical and Experimental Advances in Treatment of Visceral Leishmaniasis Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2185 - 2197. [Full Text] [PDF]
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W. I. Khan, P. A. Blennerhassett, Y. Deng, J. Gauldie, B. A. Vallance, and S. M. Collins IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice Am J Physiol Gastrointest Liver Physiol, July 1, 2001; 281(1): G102 - G110. [Abstract] [Full Text] [PDF]
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H.-W. Chen, C.-H. Pan, H.-W. Huan, M.-Y. Liau, J.-R. Chiang, and M.-H. Tao Suppression of Immune Response and Protective Immunity to a Japanese Encephalitis Virus DNA Vaccine by Coadministration of an IL-12-Expressing Plasmid J. Immunol., June 15, 2001; 166(12): 7419 - 7426. [Abstract] [Full Text] [PDF]
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 F. Dieli, G. Sireci, D. Russo, M. Taniguchi, J. Ivanyi, C. Fernandez, M. Troye-Blomberg, G. De Leo, and A. Salerno Resistance of Natural Killer T Cell-Deficient Mice to Systemic Shwartzman Reaction J. Exp. Med., December 4, 2000; 192(11): 1645 - 1652. [Abstract] [Full Text] [PDF]
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 W. C. Russell Update on adenovirus and its vectors J. Gen. Virol., November 1, 2000; 81(11): 2573 - 2604. [Full Text]
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E. Maverakis, J. T. Beech, S. S. Wilson, A. Quinn, B. Pedersen, and E. E. Sercarz T Cell Receptor Complementarity Determining Region 3 Length Analysis Reveals the Absence of a Characteristic Public T Cell Repertoire in Neonatal Tolerance: The Response in the "Tolerant" Mouse within the Residual Repertoire Is Quantitatively Similar but Qualitatively Different J. Exp. Med., February 21, 2000; 191(4): 695 - 702. [Abstract] [Full Text] [PDF]
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 V. Julia and N. Glaichenhaus CD4+ T Cells Which React to the Leishmania major LACK Antigen Rapidly Secrete Interleukin-4 and Are Detrimental to the Host in Resistant B10.D2 Mice Infect. Immun., July 1, 1999; 67(7): 3641 - 3644. [Abstract] [Full Text] [PDF]
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