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Laboratory of Experimental Immunology, Division of Basic Sciences;
Department of Experimental Transplantation and Immunology, Division of Clinical Sciences; and
Intramural Research Support Program, Science Applications International Corp., National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
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Pediatric Oncology Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892; and
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EntreMed, Inc., Rockville, MD 20850
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN--inducible Mig genes. Both
IL-12 p70 heterodimer and IFN- proteins were documented in the serum
within 24 h after intradermal injection of the
pIL-12o- plasmid, which also induced the highest level of
NK activity in the spleen and liver among the IL-12 constructs.
Interestingly, both p40 mRNA expression at the injection site and serum
protein levels followed a biphasic pattern of expression, with peaks on
days 1 and 5. Subsequent studies revealed that the ability of
intradermally injected pIL-12o- to augment NK lytic
activity was prevented by administration of a neutralizing
anti-IL-12 mAb. Finally, injection of the pIL-12o-
into BALB/c IL-12 p40-/- mice also resulted in a biphasic
pattern of IL-12 p70 appearance in the serum, and induced IFN-
protein and activated NK lytic activity in liver and
spleen. These results demonstrate that injection of delivered naked DNA
encoding the IL-12 gene mediates the biphasic systemic production of
IL-12-inducible genes and augments the cytotoxic function of NK cells
in lymphoid and parenchymal organs as a direct result of transgene
expression. The results also suggest that these naked DNA plasmids may
be useful adjuvants for vaccines against infectious and neoplastic
diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent antiangiogenic
activity (11, 12). These diverse biological effects make
IL-12 an attractive candidate as a therapeutic agent for cancer and
infectious diseases. Systemic administration of IL-12 protein alone
(2, 13, 14, 15) or in combination with IL-2
(16, 17, 18) significantly suppressed the growth of a variety
of established mouse tumors and prolonged the survival of tumor-bearing
mice. IL-12 also has efficacy as an adjuvant for vaccination against
cancer (19), and intratumoral delivery of adeno- or
retroviruses containing the IL-12 gene can cause regression of some
established tumors in mice (20, 21, 22, 23). The targeted
inactivation of both alleles of the IL-12 p40 gene impairs the
production of IFN- and the induction of a delayed-type
hypersensitivity response (24) and renders mice
susceptible to infection by Leishmania major
(24). Despite these interesting therapeutic implications for IL-12, the best approaches for delivery of IL-12 in vivo remain to be determined. Most studies have been performed using systemic delivery of the rIL-12 protein. Although the pharmacodynamics of IL-12 are more favorable than those of many other cytokines, repeated administration on a daily basis is required for maximal therapeutic activity in mice (25, 26). In addition, the repeated bolus administration of the recombinant proteins can cause undesirable side effects (27, 28). Alternative approaches for IL-12 delivery also have some limitations. For example, virus-mediated gene delivery can result in the subsequent generation of neutralizing Ab, which limits the duration that active immunotherapy is effective. In the case of cytokine-mediated retroviruses, integration of the virus genome into host chromosomes may be a concern for other deleterious effects.
The direct in vivo transfer of DNA without any carrier agents (referred to as naked DNA) was first described in 1990 as a novel form of gene therapy (29). The initial studies showed that muscle was a suitable target tissue for gene delivery (29, 30), but skin also was shown to be suitable as an alternative site for injection (31, 32, 33, 34). Some initial success of naked DNA encoding therapeutic proteins was documented by the induction of a host immune response against several infectious agents (31, 35, 36). Subsequently, naked DNA was also proven to induce local or systemic biological effects in vivo, including improvement of anemia by in vivo delivery of the erythropoietin gene (37) or recruitment of neutrophils into the site of IL-8 plasmid DNA injection (32). Major advantages for the in vivo use of highly pure plasmids include the relatively simple and inexpensive production compared with protein and the possibility that more chronic production may decrease the need for high systemic protein levels associated with bolus administration of cytokines, thereby reducing unfavorable side effects.
In this study we constructed several IL-12 expression vectors that
encode both murine p35 and p40. These plasmids were injected
intradermally (i.d.)4
and shown to induce IL-12 mediated biological activity, including
activation of NK cells, induction of IFN-, and the IFN--inducible
chemokine monokine induced by IFN- (MIG). These effects also were
detectable in IL-12 p40-/- mice, showing that
they were not mediated by endogenous production of normal host IL-12 or
contaminating endotoxin. Overall, these results show that in vivo
transferred pIL-12 DNA can induce the expected systemic bioactivities
of IL-12 and suggest that this form of gene therapy is efficient and
safe for IL-12 delivery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogen-free female BALB/c mice between 6–8 wk of age were obtained from the Animal Production Area, National Cancer Institute-Frederick Cancer Research and Development Center. These mice were housed under specific pathogen-free conditions and provided sterilized mouse chow and water ad libitum. BALB/c IL-12 p40-/- mice (38) were donated by Dr. Jean Magram, Hoffmann-La Roche (Nutley, NJ) and maintained as a small breeding colony in our own animal facility. Animal care was provided in accordance with the procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Cell lines and reagents
The hybridoma C17.8 for anti-mouse IL-12 p70 was a gift from Dr. Giorgio Trinchieri (Wistar Institute of Anatomy and Biology, Philadelphia, PA). YAC-1 and P815 cells were maintained in vitro in RPMI 1640 containing 10% FCS and 2 mM glutamine.
Plasmids
The expression vector CMV-ß, which encodes the
ß-galactosidase gene, was obtained from Clontech (Palo Alto, CA).
Four constructs of murine pIL-12 were generated as follows. First, the
pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) was modified by inserting
an SV40 intron between the CMV enhancer/promoter and multicloning site.
As shown in Fig. 1
, the p35 and p40
subunits of murine IL-12 were each driven by a separate CMV promoter,
and individual expression cassettes were oriented in either the same or
opposite directions in a single retroviral vector, except in the case
of the pIL-12 IRES plasmid, where the p40 sequence was driven by the
IRES. For some plasmids, the neomycin expression cassette was removed.
Plasmids were prepared using the Qiagen Endofree Buffer kit and
Qiagen-tip 2500 (Qiagen, Valencia, CA) and dissolved in PBS. The
endotoxin levels of the prepared plasmids were <0.05 EU/µg of DNA by
limulus amoebocyte lystate test (BioWhittaker, Walkersville,
MD).
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Fifty micrograms of plasmid DNA in 100 µl of PBS was injected i.d. at the base of the tail of the mice using a 30-gauge needle and a 1-ml syringe. For i.v. delivery, DNA was dissolved in PBS containing 5% glucose in a total volume of 200 µl and injected via the lateral tail veins.
ß-Galactosidase staining
After i.d. injection of CMV-ß as described above, the site of gene transfer was harvested at 24 h and fixed in PBS containing 2% formaldehyde and 0.2% glutaraldehyde. The skin was then stained with Bluo-gal (Life Technologies, Gaithersburg, MD) as described previously (32). Briefly, tissue was rinsed in PBS and incubated in PBS containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, and 1 mg/ml Bluo-gal (Life Technologies) at room temperature overnight. On the next day, the tissue was rinsed with PBS and postfixed in 4% formaldehyde, 100 mM sodium phosphate, and 10% methanol.
Detection and quantification of murine IL-12 and IFN- by ELISA
Blood was collected at various times from mice after injection
of plasmid DNA, and the serum was assayed for murine IL-12 and IFN-
by ELISA kits purchased from Endogen (Woburn, MA) and R&D Systems
(Minneapolis, MN), respectively.
Assessment of NK cell activity in leukocytes isolated from liver and spleen
At various times after plasmid injection, mice were euthanized, and blood, liver, and spleen were harvested. The livers were perfused with HBSS, and mononuclear cells were prepared as previously described (18). Briefly, three or four livers were dissociated on a stomacher (Tekmar, Cincinnati, OH) and centrifuged at 500 x g. The resuspended pellet was filtered with nylon gauze, overlaid on Lympholyte M (Ceder Lane Laboratories, Ontario, Canada), and centrifuged at 2600 x g for 30 min. The leukocyte layer was recovered, and the cells were washed and counted. Various numbers of leukocytes were then cocultured for 4 h with 1 x 10 451Cr-labeled YAC-1 or P815 target cells in 96-well microplates.
RT-PCR
Total RNA was prepared from snap-frozen spleens using Trizol
(Life Technologies). cDNA were synthesized with Moloney murine leukemia
virus reverse transcriptase (Promega, Madison, WI) primed with an
oligo(dT)12–18 primer (Pharmacia, Piscataway,
NJ) in the presence of 0.2 mM dNTP and 10 U of RNase inhibitor
(Pharmacia). cDNA (250 ng) was used to amplify the IFN- and Mig
genes. The reaction was performed as 30 cycles at 94°C for 30 s,
55°C for 60 s, and 72°C for 60 s. The sequences for
primers are: IFN- sense, 5'-TGCGGCCTAGCTCTGAGACAATGA3'; IFN-
antisense, 5'-TGAATGCTTGGCGCTGGACCTGTG-3'; Mig sense,
5'-GATCAAACCTGCCTAGATCC-3'; Mig antisense,
5'-GGCTGTGTAGAACACAGAGT-3'; actin sense,
5'-CAGCTGAGAGGGAAATCGTG-3'; and actin antisense,
5'-ACTGTGTTGGCATAGAGGTC-3'. Ten microliters of PCR product was resolved
in a 1.5% agarose gel along with a 100-bp m.w. marker (Life
Technologies).
Northern blot analysis
Total RNA was prepared at various times as described above from the skin at the site where DNA was injected. Five micrograms of total RNA was run in 1% agarose gels containing 1x MOPS buffer and transferred to a nylon membrane Hybond N (Amersham, Arlington Heights, IL). The RNA blot was prehybridized in Easy Hyb (Boehringer Mannheim, Indianapolis, IN) and hybridized overnight in the same buffer containing 100 ng/ml of digoxygenin-labeled murine IL-12 p40 antisense riboprobe. The membrane was then washed twice with 0.1% SDS and 0.1x SSC at 65°C for 15 min, and chemiluminescence was detected with CSPD as a substrate according to the manufacturer’s handbook (The DIG System User’s Guide for Filter Hybridization, Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm that i.d. injected DNA actually leads to expressed
genes in vivo, we initially investigated the expression of the i.d.
injected ß-galactosidase gene, CMV-ß, as a model system.
Twenty-four hours after the i.d. injection of 20 µg of CMV-ß,
ß-galactosidase activity was readily demonstrated at the site of
injection (data not shown). Histological analysis of the site revealed
the activity was mainly in the smooth muscle of the s.c. tissue (Fig. 2).
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These studies were performed to compare the abilities of the
various IL-12 plasmids to augment mouse NK activity in vitro. In
preliminary screening studies, leukocytes were isolated from liver and
spleen 4 days after the i.d. injection of control or IL-12 expression
vectors, and NK-mediated lytic activity was measured against YAC-1
target cells. In these studies pIL-12o+, in which
the p35 and p40 expression cassettes were placed in opposite
orientations in a single retroviral vector (Fig. 1
) gave the highest NK
cell activity in the liver and spleen, followed by pIL-12 IRES,
pIL-12s-, and pIL-12s+ in
the order of NK activity induced (data not shown). The results
suggested that the use of separate expression cassettes for p40 and p35
produced higher NK cell activity than that obtained employing the IRES
strategy, particularly when the p40 and p35 expression cassettes were
placed in opposite orientations. The results also suggested that the
presence of the neomycin expression cassette may have some inhibitory
effect on IL-12 production and NK activity in vivo. Therefore we
constructed the pIL-12o- in which the p35 and
p40 genes were oriented in the opposite direction from each other and
where both were under separate control of CMV promoters, but the
neomycin gene expression cassette was deleted. The study shown in Fig. 3 compares the NK-augmenting effects of
the neo- and neo+
constructs. NK lytic activity induced by
pIL-12o- was comparable to that obtained with
IL-12o+ and was 4- and 3-fold higher than levels
induced by the IRES vector in the liver and spleen, respectively.
Because there was no apparent benefit to expressing neomycin at the
site of gene delivery, the IL-12o- plasmid was
selected for further study to exclude any concerns about whether any
observed in vivo biological effects could be related to the expression
of neomycin in vivo. None of the mice that received single injections
of either pcDNA control vector or the pIL-12 constructs showed any
gross toxicities.
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and Mig genes in the spleen
Because many of the biological effects of IL-12 are known to be
mediated via induction of IFN- and subsequent induction of other
IFN--inducible genes, we studied the inducibility of the IFN- and
Mig genes. Portions of the spleens were obtained from mice treated with
the pIL-12o- and/or
pIL-12o+ plasmids alone or with pcDNA vector.
Twenty-four hours after the i.d. injection of
pIL-12o-, modest induction of IFN- gene
expression was demonstrated in the spleen by RT-PCR (Fig. 4A). Mig gene expression also
was observed in the spleen (Fig. 4B) 4 days after the
injection of pIL-12o+ and
pIL-12o-.
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The previous data demonstrated that NK cell activity was induced
by pIL-12o- by day 4. However, it was unclear
whether this was the optimal time for detecting augmented NK activity
and how this related to the production of IL-12. Therefore, a more
detailed kinetic evaluation of these events was performed at various
times between 1–14 days after i.d. plasmid injection using the same
dose of DNA described above. As shown in Fig. 5 under this time scheme, only at 5 days
after pIL-12o- injection was there a clear
increase in NK activity in the liver and spleen.
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depict the kinetics of
IL-12 and IFN- in sera pooled from three mice per group at various
times after injection of 50 µg of pIL-12o- or
pcDNA. As expected, the serum levels of these two cytokines peaked well
before the detectable augmentation of NK activity (Fig. 5).
Specifically, there was detectable IL-12 (Fig. 6A) and
IFN- protein (Fig. 6B) by 24 h after DNA injection.
Surprisingly, the IL-12 protein rose again 96 h after injection,
thereby displaying a biphasic profile of IL-12 p70 protein in the serum
after the single i.d. injection of pIL-12o-.
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To investigate the mechanism for the biphasic production of IL-12
protein, tissue was harvested from the injection site at various times
after the injection of pIL-12o- and analyzed by
Northern blot for p40 gene expression (Fig. 7). These results showed that by 24
h there was a pronounced induction of the p40 gene, which was then
down-regulated by day 3 and re-elevated on day 5, consistent with the
protein data presented in Fig. 6A above. The gene
re-expression initially detected on day 5 remained until day 7. No p40
expression was detected after pcDNA injection, demonstrating that the
gene expression observed above was directly due to transcription of the
injected pIL-12o-. Overall, these results
suggest that the biphasic expression of IL-12 protein occurred because
of a biphasic expression of the IL-12 gene.
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To confirm that the systemic NK activity elicited by
pIL-12o- was directly dependent on the
production of IL-12 protein, mice were pretreated i.p. with 25 µg of
either C17.8 rat mAb, which specifically neutralizes the murine IL-12
p70 heterodimer, or control rat Ab beginning 24 h before the
injection of 50 µg of pIL-12o- or pcDNA
injection. Spleens and livers were then harvested, and isolated
leukocytes were tested for NK activity. In this experiment, because of
a limited availability of leukocytes from the liver, a single E:T cell
ratio was employed to test NK activity, while the assay for
splenic NK activity was conducted as in the previous experiment. Mice
treated with control Ab and pIL-12o- exhibited
the expected increase in NK activity, while those treated with C17.8 Ab
and pIL-12o- showed no augmentation of NK
activity (Fig. 8).
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The data presented above cumulatively support the ability of a
locally injected IL-12 expression plasmid to induce systemically
detectable cytokine levels and biological effects. However, it is
possible that direct localization of the expression plasmid in major
organs and the focused production of IL-12 in such sites might have
more potent biological effects in specific organs. Therefore, we
investigated whether i.v. injected plasmid DNA could augment NK
activity in the spleen and liver. This approach resulted in very potent
induction of NK activity by 2 days after iv injection of the
IL-12o- plasmid (Fig. 9). This augmented NK activity was
retained at 96 h, and the levels of NK activity achieved in the
liver were quite high compared with those observed after i.d. injection
(Fig. 3).
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To completely exclude the possibility that any of the biological
effects outlined above were due to the induction of endogenous IL-12
production rather than to direct transcription and translation of the
gene product of the injected plasmid, we repeated the NK augmentation
studies in IL-12 p40-/- mice. After i.d.
injection of 50 µg of pIL-12o- into the BALB/c
p40-/- mice, IL-12 p70 was detected in the
serum by 24 h (Fig. 10A). IFN- also was
demonstrated in the serum by 24 h (Fig. 10B), and as
seen in normal mice, the effect was biphasic, showing high levels at
24 h, a decline, and subsequent re-elevation by 4 days. This
IFN- induction was confirmed by RT-PCR of spleen from the same
treatment mice (Fig. 10C). The
pIL-12o- also augmented NK activity in the
spleens of the p40-/- mice as previously
observed in normal mice (Fig. 11) by
day 4. However, in contrast to results in normal mice (Fig. 3),
augmentation of NK activity in p40-/- mice
occurred more rapidly (by 24 h) and persisted for 4 days. These
results demonstrate that the injection of an appropriately constructed
pIL-12 naked DNA plasmid can directly contribute enough IL-12 protein
to mediate potent systemic production of IL-12-inducible genes and
augment the cytotoxic function of NK cells in lymphoid and parenchymal
organ sites.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression and protein production as the
parameters of choice for detecting systemic IL-12-induced
bioactivities. We found that pIL12o+, in which
the p40 and p35 subunits were driven by a separate CMV
promoter/enhancer and where the expression cassettes were oriented in
the opposite direction in the same construct, produced the highest NK
cell activity in the liver and spleen. The degree of systemic
augmentation of NK activity was higher than that achieved with other
plasmids, including those in which p40 and p35 were both in the same
orientation but driven by individual CMV promoter/enhancers or using an
IRES plasmid in which the expression cassette was in a tandem
orientation. Some previous reports also suggested that the coexistence
of CMV enhancer and SV40 promoter for neomycin expression decreased
gene expression from the CMV enhancer (41). Therefore, in
an attempt to obtain the most efficient in vivo construct possible,
IL-12o-, the neomycin expression cassette was
removed from the pIL-12o+ plasmid, and this
pIL-12o- demonstrated NK activity at least
comparable with that obtained using pIL-12o+. In
addition, pIL-12o- was superior to the
pIL-12o+ construct for induction of IFN- in
vivo (data not shown). These results extend previous studies
(34) that reported an augmentation of splenic NK activity
by i.d. injection of IL-12 cDNA by demonstrating that the pIL-12 also
can augment NK activity in the liver.
The best cellular target for in vivo transfection remains unclear. In
our initial experiments we chose the i.d. route to deliver naked DNA
for two reasons. First, it is technically simpler than i.m. injection,
and second, the previous literature demonstrates that i.d. transferred
genes are expressed more quickly than those expressed after injection
into muscle. Because we also plan to contrast naked DNA delivery with
protein therapy for the treatment of rapidly growing established tumors
in mice, we speculate that a process that results in more rapid gene
expression in vivo could be more beneficial than one where initial gene
expression is more prolonged, but delayed in its onset. As shown in
Fig. 2, the injected DNA was expressed mainly in the i.d. smooth
muscle, while in previous studies the activity of the i.d. injected
gene product was visualized predominantly in the epidermis in the human
and pig skin tissue (32). Thus, there may be a preference
for a particular cell type for the DNA entry depending on the nature of
the construct, the genes to be expressed, or the species to be
injected. By Northern blot analysis, p40 gene expression was detectable
up to 7 days at the site of the pIL-12o-
injection, and mRNA expression was highest at 24 h, with another
peak appearing 5 days later after a single injection of plasmid. Serum
levels for IL-12 after pIL-12o- injection
paralleled the kinetics of this p40 gene expression, supporting the
observed biphasic nature of p40 gene expression. This biphasic pattern
of expression was surprising, and we speculated that the first peak
represented IL-12 produced from the injected plasmid, while the second
peak could be due to subsequent production of IFN- by T and NK cells
(42) and subsequently induction of more IL-12 production
by phagocytic cells (43). However, this is not the case,
because the same biphasic gene expression is obtained after
pIL-12o- injection into
p40-/- mice, in which endogenous IL-12 p70
cannot be induced. Taking these three independent results (p40 Northern
blotting, IL-12 and IFN- serum ELISAs in normal mice and
p40-/- mice) together, therefore, we conclude
that i.d. pIL-12o- injection results in a
biphasic expression pattern for the transferred gene itself in vivo.
Although previous studies using i.m. DNA injection revealed a gradual
increase in protein expression by the transgene up to 14 days, with
activity detected as long as 120 days (29, 44), we know of
no reports of clear biphasic or intermittent expression. To date, the
relatively short term (e.g., 1–7 days) kinetics of naked DNA in vivo
have not been investigated in complete detail, and we plan to examine
the mechanism for these biphasic effects.
Taking into consideration the expression data outlined above, it may
not be surprising that the highest NK cell activity in liver and spleen
was not observed until about 4 days after
pIL-12o- injection (i.d.) even though the blood
level of IL-12 was highest at about 24 h. Recent data from our
laboratory have shown that a single administration of recombinant mouse
IL-12 protein (0.5 µg/day) induced the highest NK activity in the
liver compared with daily injection for 2–4 days (45). In
these studies a decrease in NK activity after repeated administration
of IL-12 was accompanied by a reduced number of NK cells. However, in
the studies using the pIL-12o- no decrease in
the number of NK cells in the liver was observed even 4 days after
pIL-12o- injection (data not shown), suggesting
a basic biological difference in the regulation of hepatic NK cells by
exogenous IL-12 protein vs pIL-12o-.
Interestingly, we noted in our previous studies with IL-12 protein
administration that the ability of IL-12 to induce recruitment of NK
cells to the liver is dependent on the production of IFN-
(45). Therefore, IFN- induced by IL-12 may contribute
to a recruitment of NK cells to at least some sites. In addition, the
IFN--inducible Mig gene, which serves as another indicator of
systemic effects of IL-12, also was induced. Although this expression
of Mig may have no direct relevance to induction of Th1 responses, it
may play some role in the IFN--dependent recruitment of NK cells
induced by IL-12 (45). We are currently studying the role
of IFN--inducible genes in IL-12-induced leukocyte recruitment.
Another intriguing finding of our study is that in
p40-/- mice, injection of
pIL-12o- induced more potent augmentation of NK
activity and induced more IFN- than in normal mice, particularly in
the second peak. The controlled production of the p40 subunit that is
usually produced in large excess over the p35 subunit and can
antagonize the biological effects of IL-12 p70 may explain the higher
responsiveness to IL-12 translated from the expressed
pIL-12o- in p40-/-
mice.
In an effort to further optimize and understand the immunomodulatory potential of the pIL-12o-, naked DNA delivery by the i.v. route also was investigated. A previous report found that the i.v. injection of naked DNA in PBS resulted in degradation within 5 min and the absence of any protein expression in various organs (46). However, in our studies i.v. injection of 200 µg of pIL-12o- effectively induced NK activity in both liver and spleen by 2 days after injection, and the augmented NK activity remained detectable at 4 days. Consistent with this observation are studies by Wang et al., who reported kallikrein gene expression in heart, lung, and liver even 3 wk after a single i.v. injection of 500 µg of DNA dissolved in PBS containing 5% glucose (47). Thus, the addition of 5% glucose, as used in our studies, may be useful for stabilizing the DNA for i.v. injection, although the mechanism involved in the stability has not been determined yet.
In vivo delivery of plasmid DNA is becoming more commonly used as a
novel vaccination method (48), where i.m. or i.d.
injection of Ag-coding DNA favors the effective development of Th1
responses (49), and a previous study has demonstrated that
the injection of IL-12 cDNA can actually delay the growth of
subsequently injected murine renal cancer cells. A recent finding also
has been reported that particular unmethylated DNA sequences with CpG
motifs preferentially stimulate the production of IFN-, IL-12, and
IL-18 (50) and can activate various effector leukocyte
cells, including NK cells. In the bacterial genome these sequences are
often unmethylated and 20 times as common as in mammalian DNA, whose
CpG motif is methylated in >80% of the cases. Thus, some mammalian
DNA and, more preferentially, bacterial DNA also may target induction
of a Th1 response by host leukocytes. In contrast, other approaches,
such as DNA vaccination by the gene gun, preferentially induce Th2
responses (51). Although the gene gun requires 1/100th
less DNA (usually 0.5–2 µg) compared with i.d. or i.m. DNA (50–200
µg) vaccination to elicit biological effects, it also is possible
that the amount of DNA delivered by the gene gun technology is not
sufficient to effectively trigger Th1 immune responses locally or
systemically. Our experiments in normal and
p40-/- mice demonstrate clearly that the i.d.
injection of pIL-12o- DNA triggers a strong Th1
response that is independent of a secondary IFN--induced or
DNA-nonspecific production of endogenous IL-12. Thus, the i.d. delivery
of pIL-12o- leads to effective transcription and
translation of biologically active IL-12 p70 that systemically induces
cytokines and host effector cell functions.
The effective induction of a Th1 response, as indicated by IFN-
synthesis following pIL-12o- in our studies, may
be a major advantage for the therapeutic use of IL-12 in vaccine
approaches to infectious disease and cancer treatment. Ghosh et al.
reported that T cells from mice bearing tumors for >1 mo gradually
lose the Th1 phenotype (52), and the reversal or
prevention of this effect may be important for maximizing the response
to therapeutic vaccines. In addition to any systemic effects, the
amount of IL-12 available at the tumor site contributes to both the
type and the number of infiltrating leukocytes and the events leading
to tumor regression (53). As for infection, a similar
effect has been confirmed during vaccination against Schistosoma
mansoni, in which vaccination with eggs and IL-12 prevents the
subsequent pulmonary granuloma formation and tissue fibrosis that are
associated with a Th2-dominated pattern of cytokine expression
(54, 55). Also, as mentioned previously, IL-12 plays an
important role in protecting against infection by L. major
(24).
Overall, the IL-12 gene delivery approach described herein demonstrates the potency of appropriate DNA expression plasmids for the induction of systemic and local Th1-type responses. Such effects, in the absence of the practical limitations often observed during the use of viral vectors as gene delivery systems, suggest the considerable utility of this approach for vaccine-based prophylactic or therapeutic strategies in the treatment or prevention of infections and neoplastic diseases.
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Acknowledgments |
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ELISAs. We
thank Dr. Kristin Komschlies for advice in the production of mAbs. We
acknowledge Susan Charbonneau and Joyce Vincent for excellent
manuscript preparation and editorial assistance. |
Footnotes |
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2 Current address: Greenebaum Cancer Center, Room 7-023, Brassler Research Building, 655 West Baltimore Street, University of Maryland Medical School, Baltimore, MD 21201.
3 Address correspondence and reprint requests to Dr. Robert H. Wiltrout, Experimental Therapeutics Section, Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address:
4 Abbreviations used in this paper: i.d., intradermal(ly); MIG, monokine induced by IFN-; IRES, internal ribosomal entry site.
Received for publication February 22, 1999. Accepted for publication May 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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