| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Division of Cancer Research and
Division of Clinical Research, Institute of Biomedical Sciences, Academia Sinica;
Graduate Institute of Life Sciences, National Defense Medical Center;
§
Graduate Institute of Clinical Medicine and
¶
Graduate Institute of Microbiology, National Taiwan University; and
||
Development Center for Biotechnology, Taipei, Taiwan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene exhibited a significant enhancement of Th1 cells and increased
production of anti-HBV surface IgG2a Ab, as well as a marked
inhibition of Th2 cells and decreased production of IgG1 Ab. In
contrast, coinjection of the IL-4 gene significantly enhanced the
development of specific Th2 cells and increased production of IgG1 Ab,
whereas Th1 differentiation and IgG2a production were suppressed.
Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced
the development of Th1 cells, while the development of Th2 cells was
not affected, and the production of IgG1 and IgG2a Ab were both
increased. The CTL activity induced by HBV DNA vaccination was most
significantly enhanced by codelivery of the IL-12 or IFN- gene,
followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas
codelivery of the IL-4 gene suppressed the activity. When challenged
with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant
reduction of tumor growth was observed in mice that were coadministered
the IL-12 gene but not the IL-4 gene. Taken together, these results
demonstrate that application of a cytokine gene in a DNA vaccine
formulation can influence the differentiation of Th cells as well as
the nature of an immune response and may thus provide a strategy to
improve its prophylactic and therapeutic efficacy. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The protective value of the immune response is highly dependent upon
the types of cytokines produced by CD4+ Th cells. This
concept was first clearly demonstrated in the in vivo study of the
immune response to Leishmania major infection in
a murine model, in which resistance or susceptibility to the parasite
was dependent on the development of respective Th1 or Th2 effector
cells (26, 27). Th1 cells that produce IL-2 and IFN- induce
activation of macrophages, delayed-type hypersensitivity, and
production of IgG2a Ab (28, 29). In contrast, Th2 cells that
predominantly produce IL-4, IL-5, IL-10, and IL-13 promote development
of eosinophilia as well as generation of Abs of IgG1 and IgE isotype
(28, 29). Indeed, Th1 and Th2 cells constitute a dynamic and mutually
inhibitory network that tends to support distinct elements of the
immune system. The best defined among the stimuli that direct the
development of naive CD4 cells into Th1 or Th2 effectors are the
cytokines to which the naive cells are exposed during antigenic
stimulation. Thus, IL-4 plays a critical role in the priming of Th2
cells (30), whereas IL-12 alone (31) or together with IFN- (32)
stimulates differentiation of naive T cells into the Th1
lymphokine-producing phenotype.
We have shown previously that coexpression of IL-2 and hepatitis B
virus (HBV)3 envelope protein
within the same plasmid vector resulted in a dramatic increase in its
ability to induce humoral and cellular immune responses to HBsAg (33).
The IL-2 adjuvant activity also helps the HBV DNA vaccine elicit high
anti-HBs titers in animals that usually fail to respond to rHBsAg
vaccination, a phenomenon that is closely related to certain MHC
haplotype (34, 35). In addition, splenocytes derived from mice that
received plasmids coexpressing IL-2 and the HBV envelope protein
produced much stronger Th1-like responses than those from mice that had
been inoculated with plasmids encoding the envelope protein alone. In
the present report, we investigate whether coadministration of other
cytokine genes is able to modulate the immune response by favoring the
development of Th1 vs Th2 cells. We found that mice given injections of
the plasmid encoding HBsAg together with plasmids encoding IL-12 or
IFN- promoted Th1 and suppressed Th2 differentiation. Conversely,
coinjection of a plasmid expressing IL-4 promoted Th2 and suppressed
Th1 differentiation. Coadministration of the IL-2 or GM-CSF gene mainly
enhanced Th1 cell differentiation, and left Th2 cell development
unaffected. In accordance with the different Th populations induced by
DNA vaccination under influence of various cytokine genes, the Ab
isotype, CTL activity, as well as the in vivo antitumor effect, were
also substantially altered.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The plasmid vector pS encoding HBV major envelope proteins was
constructed previously (33). This eukaryotic expression vector was
modified from plasmid pcDNA3 (Invitrogen, San Diego, CA) containing the
CMV early promoter/enhancer sequence and the polyadenylation and 3'
splicing signals from bovine growth hormone. The cDNA of murine
IFN-, IL-4, and GM-CSF were obtained by reverse transcription and
PCR amplification of RNA derived from about 1 x
108 mouse (C3H/HeN) splenocytes stimulated for 4
h, 8 h, and 24 h, respectively, with 5 ng/ml of PMA (Sigma
Chemical Co., St. Louis, MO) and 5 µg/ml of Con A (Sigma) in culture
medium. The fragment containing the murine IL-2 coding sequence was
generated by PCR from plasmid pmut-1 (ATCC 37553; American Type Culture
Collection (ATCC), Rockville, MD). The upstream PCR primer for each
cytokine gene contains a BamHI site, and the downstream
primer contains an EcoRI site. PCR products of IL-2, IL-4,
GM-CSF, and IFN- were digested with BamHI and
EcoRI, gel purified, and inserted between the
BamHI and EcoRI sites of pcDNA3 to generate
pIL-2, pIL-4, pGM, and pIFN-, respectively. The fragments containing
the p35 and p40 coding sequences of murine IL-12 were generated by PCR
from plasmids BLpSV35 and BLpSV40 (kindly provided by Michale T. Lotze,
University of Pittsburgh, Pittsburgh, PA), respectively, and cloned
into a bicistronic plasmid, pTCAE (33), under the control of
discrete CMV promoters. Plasmid DNA was purified from transformed
Erichia coli strain DH5
by Qiagen Plasmid Giga Kits
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions
and stored at -70°C as pellets. The DNA was reconstituted in sterile
saline at a concentration of 1 mg/ml for experimental use.
Cell transfection and cytokine gene expression
The expression of cytokine genes by the plasmid DNA was performed in a transient transfection assay using Lipofectamine (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. Briefly, C2C12 mouse myoblasts (1 x 105 cells; ATCC 1772) were cultured in DMEM plus 10% FCS (DMEM-10) in a six-well tissue culture plate until the cells reached approximately 50 to 80% confluence. Three micrograms of plasmid DNA was mixed with 20 µl of Lipofectamine in 200 µl of OPTI-MEM medium (Life Technologies) at room temperature. Following a 20-min incubation, the DNA-liposome complexes were diluted in 800 µl of OPTI-MEM and slowly added to cells, which had been prewashed twice with 5 ml of OPTI-MEM. After a 16-h incubation, the DNA-liposome complexes were removed, 2 ml of DMEM-10 was added to each well, and incubation was continued for another 48 h. The supernatant of each well was collected and stored at -80°C for cytokine analysis.
Cytokine ELISAs and proliferation assays
The cytokine activity present in the supernatant of plasmid DNA-transfected cells was assayed for its ability to support the proliferation of appropriate responsive cells: IL-2 and IL-4 were tested on HT-2 cells (36), GM-CSF was measured on NFS-60 cells (37), and IL-12 was determined on activated lymphoblasts. The HT-2 and NFS-60 cells were grown in RPMI 1640 containing 10% FCS (RPMI 10) supplemented with 1% supernatant of transfectoma cell lines Id-IL-2 (38) and Id-MoGM (39), which produce Id-cytokine fusion proteins containing murine IL-2 and GM-CSF activity, respectively. To perform the proliferation assay, samples were added in triplicate to 96-well plates in RPMI 10 with 5,000 HT-2 or NFS-60 cells to a total volume of 0.1 ml and incubated in a humidified incubator for 16 to 24 h at 37°C and 5% CO2. IL-12 proliferation assay was performed as previously described (40). Briefly, the Con A-activated lymphoblasts were prepared by culturing C57BL/6 splenocytes in TCM medium (Celos Laboratory, Hopkins, MN) containing 2% FCS, 20 ng/ml of human rIL-2 (Endogen Inc., Cambridge, MA) and 2 µg/ml of Con A (Sigma) at a density of 2 x 106 cells/ml for 2 to 3 days. The lymphoblast cells were then harvested and cultured at 50,000 cells/well with test samples in TCM plus 5% FCS for 24 h. After incubation, 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) was added to each well in 50 µl of growth medium, and cells were harvested 4 to 6 h later using a FilterMate (Packard, Meriden, CT) automatic cell harvester; the incorporated radioactivity was determined using TopCount microplate scintillation counter (Packard). The concentration of cytokines in the samples was determined from the standard curves generated using recombinant murine IL-2, IL-4, GM-CSF, or IL-12, all of which were purchased from PharMingen (San Diego, CA).
ELISA detection systems (PharMingen) were also used to screen for the
presence of IFN-, IL-2, and IL-4 cytokines in test samples. The
capture Abs for murine IFN-, IL-2, and IL-4 were R46A2 (rat IgG1),
JES6-1A12 (rat IgG2a), and BVD4-1D11 (rat IgG2b), respectively. The
detection biotinylated Abs for IFN-, IL-2, and IL-4 were XMG1.2 (rat
IgG1), JES6-5H4 (rat IgG2b), and BVD6-24G2 (rat IgG1), respectively.
Briefly, microtiter plates were coated with 50 µl of
anti-cytokine capture mAb at the concentration of 2 µg/ml
overnight at 4°C. The plates were washed twice with PBS/Tween and
blocked with 200 µl per well of 10% bovine calf serum in PBS for
2 h at room temperature. Then, the plates were washed twice and
incubated with duplicates of serially diluted samples and standards
overnight at 4°C. One hundred microliters of the biotinylated
anti-cytokine mAb at a concentration of 2 µg/ml was added to each
well and incubated at room temperature for 1 h. The plates were
then washed six times, 100 µl of avidin-phosphatase (1:500;
PharMingen) was added, and the mixture was incubated at room
temperature for 30 min. Following multiple final washings, the color
was developed with p-nitrophenyl phosphate (Sigma) as the
substrate, and absorbance at 405 nm was measured using an ELISA plate
reader. The concentration of cytokines in the samples was determined
from the standard curve.
Immunization of mice
Female BALB/c (H-2d) and C57BL/6
(H-2b) mice were obtained form the Laboratory Animal
Facility, Institute of Biomedical Sciences, Academia Sinica, Taipei,
Taiwan. All mice were immunized at 6 to 8 wk of age as described
previously (33). In brief, groups of three to five mice were
anesthetized and injected i.m. in the left hind thigh muscle with a
mixture of 100 µg of pS and 100 µg of pcDNA3, pIL-2, pIL-4, pGM,
pIFN-, or pIL-12. Mice immunized with 200 µg of pcDNA3 served as
negative controls. In some experiments, mice were immunized with 100
µg of pS in the left thigh and 100 µg of each cytokine vectors or
the control plasmid in the right thigh. For protein immunization
controls, mice were given s.c. injections in the base of the tail with
4 µg of yeast-derived rHBsAg (41) in CFA (Life Technologies, Grand
Island, NY).
Ab assay
Serum samples were collected by tail bleeding at different times, beginning 1 wk after immunization, and analyzed for the presence of HBsAg-specific Abs. Microtiter plates were coated with 5 µg/ml of yeast-derived rHBsAg. After incubation with 200 µl of 5% powdered milk in PBS on each well for 1 h to prevent nonspecific binding, 50 µl of serial dilutions of test sera was added to each well and incubated overnight at 4°C. After the samples were washed with PBS, bound proteins were detected with horseradish peroxidase-conjugated goat anti-mouse IgM (1:2000, Cappel-Organon Teknika, Veedijk Belgium) or anti-mouse IgG (1:2000; Cappel). Color was generated by adding 2,2'-azino-bis(ethylbenzthiazoline sulfonic acid), and absorbance at 492 nm was measured on an ELISA reader. For measurement of IgG1 and IgG2a anti-HBs isotypes, biotin-conjugated rat anti-mouse IgG1 (1:500, PharMingen) and rat anti-mouse IgG2a (1:500, PharMingen) were used as detectors. Avidin-alkaline phosphatase (1:1000, PharMingen) was then added. Color was developed with the addition of p-nitrophenyl phosphate (Sigma), and absorbance at 405 nm was measured. Concentrations of total IgG anti-HBs Abs in serum samples were estimated from the standard curve generated by a HBsAg-specific mAb (H25B10, ATCC CRL-8017) and expressed as µg/ml for IgG. For measurement of IgM, IgG1, and IgG2a Abs, readings were referenced to a standard serum pooled from four mice given i.p. injections of 2 µg of rHBsAg with CFA and bled 4 wk after immunization. The standard curves were generated using the pooled anti-HBs sera, and results were expressed as arbitrary units per milliliter (U/ml; 1 U = 50% maximum OD).
Flow cytometric analysis
The populations of splenocytes derived from immunized animals
were analyzed by flow cytometer. Splenocytes were stained with
FITC-conjugated anti-mouse CD4 or anti-mouse CD8, combined
with phycoerythrin-conjugated anti-Thy1.2; all Abs were purchased
from PharMingen. Stained cells were analyzed on a FACSCallibur (Becton
Dickinson, Mountain View, CA).
Assays for lymphocyte proliferation and cytokine secretion
To determine whether HBsAg-specific lymphoproliferative responses were induced in immunized aimals, spleens were removed 2 wk after immunization to make single-cell suspensions. CD4--depleted and CD8--depleted immune splenocytes were prepared by immunomagnetic depletion of CD4+ and CD8+ lymphocytes, respectively, and replaced with the same number of purified CD4+ or CD8+ cells isolated from naive mice. The immunomagnetic depletion or enrichment of CD4+ and CD8+ lymphocytes was performed by a magnetic activated cell sorter (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), using microbeads conjugated with rat anti-mouse CD4 and rat anti-mouse CD8 mAbs (Miltenyi Biotec), respectively, according to the manufacturer’s instructions. To perform the lymphoproliferative assay, 100 µl of 2 x 106/ml unfractionated, CD4--depleted, or CD8--depleted splenocytes in complete RPMI 10 were added to each well in 96-well flat-bottom plates. Stimulated wells received purified rHBsAg at a concentration of 30, 10, or 1 µg/ml; transferrin (120 µg/ml, Sigma) served as a negative control Ag and Con A (5 µg/ml, Sigma) as a positive mitogenic control. Control wells received cells only. Cells in all the wells were cultured in a total volume of 200 µl of medium. After 4 days in culture, the cells were pulsed with [3H]thymidine (1 µCi/well) for 18 h and harvested with FilterMate (Packard) and the incorporated radioactivity was determined by TopCount (Packard). The stimulation index was calculated as the mean cpm of the stimulated wells divided by the mean cpm of the control wells.
To measure cytokine secretion, splenocytes were cultured as described
above with the same panel of Ags or mitogen over the same range of
concentrations with the exception that after 3 days in culture,
cell-free supernatants were harvested and assayed immediately or stored
at -80°C. These supernatant were screened for the presence of
IFN-, IL-2, and IL-4 using ELISA detection systems as described in
the previous section.
Cytotoxicity assays
BALB/c mice were immunized i.m. with 100 µg of pS and 100 µg of each of the cytokine vectors or the control plasmid. Spleens were removed 3 wk after immunization to measure the CTL activity. CD4--depleted and CD8--depleted immune splenocytes were prepared as described in the previous section. Single-cell suspensions of unfractionated, CD4--depleted or CD8--depleted splenocytes were treated with 5 ml per spleen of ACK lysis butter (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2) for 5 min at room temperature to remove RBC. HBsAg-expressing P815 cells, a murine mastocytoma line (H-2d, DBA/2 mice), and CT26 cells, a murine colon carcinoma (H-2d, BALB/c mice), were generated by permanent transfection with 20 µg of plasmid pS, selected for G418-resistant clones, and screened for the expression of HBsAg by ELISA and immunostaining. One of the HBsAg-expressing clone from each transfected cell line was selected and designated as P815/S and CT26/S, respectively. To perform the cytotoxic assay, responder splenocytes (4 x 106 per well) were first stimulated with irradiated (8000 rad) P815/S or CT26/S transfectants (4 x 106 per well) in RPMI 10 and 20 U/ml human rIL-2 (Genzyme Corp., Cambridge, MA) in 24-well plates for 4 days at 37°C. A chromium release assay was employed to measure the ability of in vitro-stimulated responder cells to lyse P815/S and CT26/S as well as the nontransfected parental cells. Target cells (1 x 106 in 0.1 ml RPMI 10) were labeled with 0.1 mCi radiolabeled sodium chromate (Amersham) in 0.1 ml normal saline for 2 h at 37°C, washed three times with RPMI 10, and resuspended at a concentration of 5 x 104 per ml in RPMI 10. A threefold serial dilution of 100 µl of stimulated responder splenocytes (starting from 5 x 105 cells) was added to individual wells containing 100 µl of labeled target cells (5000 cells). In some experiments, the anti-CD4 mAb (GK1.5, ATCC TIB-207) or anti-CD8 mAb (53-6.72, ATCC TIB-105) was added to the culture medium during the cytotoxic assay at a concentration of 10 µg/ml. After a 6-h incubation at 37°C, 100 µl of culture supernatant was collected for gamma radiation counting. The percent specific lysis was calculated as [(experimental release - spontaneous release)]/(total release - spontaneous release)] x 100. Spontaneous release represents the amount of radioactivity released from target cells without the addition of effector cells. Total release represents the amount of radioactivity released following lysis of target cells after the addition of Triton X-100 to 1.0%.
In vivo tumor protection
Three weeks after injection of plasmid pS and various cytokine genes, individual groups of mice were challenged s.c. in the left lateral flank with 1 x 105 CT26 cells or the HBsAg gene-transfected CT26/S cells. Tumor growth was measured every second or third day.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The presence of certain cytokines during antigenic stimulation has
been shown to be of decisive importance in directing the development of
naive CD4+ cells into Th1 or Th2 effectors and thus
may determine the outcome of many infectious and autoimmune diseases.
DNA vaccination is a recently developed vaccine technology and has been
reported as able to induce humoral and cellular immunities in many
different disease models. To study whether the phenotype of immune
responses to a DNA vaccine can be altered by cytokines, a previously
described plasmid vector pS (33), which encodes HBsAg, was used as a
model system. Since local expression of cytokines at the site of immune
interaction is important to mediate its function, we hypothesized that
codelivery of a cytokine vector with the HBV DNA vaccine might achieve
this purpose. The cytokine-producing vectors pIL-2, pIL-4, pGM,
pIFN-, and pIL-12 encoding murine IL-2, IL-4, GM-CSF, IFN-, and
IL-12, respectively, were constructed as described in Materials
and Methods. To test whether the various cytokine vectors can
produce biologically active cytokines, mouse C2C12 myoblasts were
transiently transfected with each of the vectors. Two days after
transfection, the amount of cytokines present in the culture
supernatant was analyzed by ELISA (for IFN-) or proliferation assays
using appropriate responsive cells (HT-2 cells for IL-2 and IL-4;
NFS-60 cells for GM-CSF and mitogen-activated lymphoblast for IL-12).
Plasmids pIL-2, pIL-4, pGM, pIFN-, and pIL-12 produced 45.8 ±
4.8, 77.6 ± 6.8, 48.4 ± 6.5, 11.3 ± 0.3, and
17.6 ± 5.3 ng/ml of cytokine proteins, respectively, each with
its particular biologic activity.
Codelivery of cytokine vectors enhances T cell proliferative responses to pS DNA vaccination
The enhancing effect of various cytokines on T cells was then
examined. Groups of C57BL/6 mice were given i.m. injections of pS
alone, or a mixture of pS plus a cytokine vector or the parental vector
pcDNA3. Mice that received pcDNA3 served as negative controls. Mice
immunized with rHBsAg were also included as protein immunization
controls. At 2 wk after immunization, splenocytes were examined for
proliferation in response to specific Ag stimulation. Both plasmid DNA
and rHBsAg immunization elicited significant proliferative responses
over a range of concentrations (1–30 µg/ml; Table I
). Coinjection of plasmids expressing
IL-2, GM-CSF, IFN-, or IL-12 with pS enhanced the cellular
proliferation by two- to threefold, while plasmid encoding IL-4 gave a
less significant increase in the proliferative response (Table I). The
enhancement in cellular proliferation by cytokine vectors cannot be
attributed simply to the intrinsic adjuvant effect of plasmid DNA as
previously reported (42), since splenic lymphocytes derived from mice
inoculated with pS plus pcDNA3 proliferated at similar levels as those
immunized with pS alone. Mice vaccinated with the control pcDNA3 vector
did not respond to HBsAg, and all mice failed to respond to transferrin
included as a control Ag, indicating that the observed T cell
proliferation was HBsAg specific. We also found that the adjuvant
effect conveyed by the cytokine vector was only observed when the DNA
vaccine was coinjected with the cytokine vector. Mice immunized with pS
on the left thigh and cytokine cDNA vectors on the right thigh did not
enhance the HBsAg-specific proliferative response (data not shown),
indicating that the presence of cytokines at the site of immune
interaction was important for this enhancement.
|
, or pIL-12 was 32.7, 31.9, 31.1, 27.9, and 33.6%,
respectively, which was comparable with that from mice immunized with
pS plus pcDNA3 (30.8%) or pcDNA3 alone (30.9%). The frequency of
CD8+ lymphocytes was also similar among groups of mice
injected with or without cytokine vectors: 13.3% (pS + pIL-2),
14.2% (pS + pIL-4), 11.7% (pS + pGM), 12.0% (pS +
pIFN-), and 13.6% (pS + pIL-12) vs 11.0% (pS + pcDNA3)
and 12.7% (pcDNA3). To determine which lymphocyte population was
responsive to HBsAg, spleen cells showing proliferative responses were
further tested by immunomagnetic depletion of CD4+ or
CD8+ lymphocytes. In all immunized groups, depletion of
CD4+ cells significantly decreased the HBsAg-specific
proliferative response, whereas depletion of CD8+ cells had
little effect on this response (Fig. 1),
indicating that the bulk of the proliferative responses could be
accounted for by CD4+ cells.
|
To establish whether the humoral responses to the HBV DNA vaccine
can be affected by simultaneous expression of cytokine genes, groups of
five mice were given i.m. injections of a mixture of pS and various
cytokine vectors. Mice receiving 4 µg of rHBsAg were also included as
a comparison. Serum from each mouse was obtained at week 4 and week 8
following DNA or protein injection for analysis of IgM and IgG
anti-HBs Ab responses, respectively. As shown in Figure 2, both plasmid pS and rHBsAg elicited
significant titers of HBsAg-specific IgM and IgG Abs. Coinjection of
IL-2, IL-4, GM-CSF, IL-12, or IFN- genes with pS all resulted in
much stronger IgM anti-HBs Ab compared with mice receiving pS plus
the parental vector (Fig. 2A). The IgG anti-HBs
Ab were enhanced two- to threefold in mice that had received pS
together with pIL-2, pIL-4, pGM, or pIL-12 compared with those
immunized with pS plus the parental vector (Fig. 2B).
In contrast, we found that mice given injections of plasmids pS and
pIFN- showed decreased IgG anti-HBs titers compared with the
control mice. We also performed kinetic studies on the DNA
vaccine-induced humoral responses and found that the adjuvant effect
conveyed by the cytokine vector was observed only at the early time
intervals, from week 4 through 20, after DNA immunization (data now
shown). The influence was subsequently decreased, and by week 35, the
difference in anti-HBs titers between groups immunized with and
without cytokine coexpression became negligible. Mice that were given
injections of pS and the cytokine vector on different thighs showed no
alterations in the anti-HBs Ab titers (data not shown), indicating
the importance of colocalization of cytokines with Ag to exert their
adjuvant effects.
|
It is known that the subsets of Th cells can be distinguished by
the pattern of cytokines that they produce. Th1 cells produce IFN-
and IL-2, and Th2 cells produce IL-4, IL-5, IL-10, and IL-13 (27, 43, 44, 45). To study the effect of cytokine coexpression on the
development of Th cells induced by DNA vaccination, cytokines profiles
released from HBsAg-stimulated splenocytes were compared among groups
of mice immunized with pS with or without cytokine gene coinjection.
Spleen cells from animals immunized with plasmid pS plus pcDNA3 or
rHBsAg produced both Th1 (IFN-, IL-2) and Th2 (IL-4) cytokines (Fig. 3). Coexpression of IL-12 or IFN- with
the HBV DNA vaccine resulted in a significant increase in Th1 cytokine
production (Fig. 3, A and B); IFN-
levels were increased six- to sevenfold, and IL-2 levels were increased
about fivefold. This was accompanied with a complete shutoff of IL-4
production (Fig. 3C). In contrast, cells from mice
coimmunized with pS and pIL-4 produced much more IL-4 and much less
IL-2 and IFN- as compared with cells from the control mice,
indicating a bias toward the Th2 immunity. Coinjection of plasmids
pIL-2 or pGM enhanced development of T cells that produce IL-2 and
IFN- in response to challenge with a specific Ag, while cells that
produce IL-4 were not significantly affected. These results indicate
that the specific Th cell populations induced by a DNA vaccine can be
altered by local expression of cytokines. Coexpression of IL-12 or
IFN- promotes Th1 and down-regulates Th2 development, coexpression
of IL-4 promotes Th2 and down-regulates Th1 development, and
coexpression of IL-2 or GM-CSF largely enhance Th1 development and to a
lesser extent Th2 development.
|
The patterns of Ab isotypes produced in response to immunization
are reliable indicators of the types of cytokines produced in vivo.
IgG2a is produced as a consequence of Th1 cell activation and IFN-
secretion, whereas IL-4 enhances the production of IgG1 and suppresses
IgG2a (46, 47). We therefore measured anti-HBs IgG isotypes in the
sera of mice treated with pS and the plasmid encoding various cytokines
at week 8 after immunization, the time point at which peak Ab titers
were achieved in vaccinated animals. As shown in Figure 4, coexpression of IL-12 or IFN-
resulted in a dramatic increase of anti-HBs IgG2a Abs, whereas
titers of HBsAg-specific IgG1 Ab were decreased, indicating enhancement
of Th1 and suppression of Th2 cell function in immunized animals. In
contrast, mice treated with plasmids pS and pIL-4 showed an inhibition
of anti-HBs IgG2a Ab, while the production of IgG1 Ab was
increased, indicating suppression of Th1 and promotion of Th2 cell
function in vivo. Animals coinjected with plasmids encoding IL-2 or
GM-CSF showed a significant, five- to sevenfold, increase of
anti-HBs IgG2a Ab, and a much lower increase of IgG1 Ab, indicating
enhancement of Th1 cell function but no significant change in Th2
cells. We also analyzed serum samples from mice given injections of pS
and the cytokine vectors at different thighs and found that the levels
of IgG1 to IgG2a isotypes were not altered (data not shown).
|
It is well known that cellular immunity is highly dependent upon
the types of cytokines produced by Th cells. Thus, we tested whether
the CTL activity induced by HBV DNA vaccine was influenced by
coexpression of cytokines. Splenocytes from mice vaccinated with
plasmids pS with or without a cytokine vector and restimulated in vitro
were analyzed for their ability to lyse syngeneic cells expressing a
transfected HBsAg gene (P815/S). Coinjection of pIL-2, pGM, pIFN-,
or pIL-12 augmented HBsAg-specific CTL activity, in which enhancement
by pIFN- or pIL-12 was consistently greater than that mediated by
pIL-2 or pGM (Fig. 5A).
In contrast, coinjection of pIL-4 substantially decreased the CTL
activity as compared with that induced by pS without cytokine
coexpression (Fig. 5A). The CTL activity was HBsAg
specific, since mice immunized with pcDNA3 alone did not induce any
detectable HBsAg-specific lysis and no CTL activity was observed in any
of the groups of mice when parental P815 cells were used as target
cells (data not shown). The effector cells that lysed HBsAg-expressing
transfectants in vitro were eliminated by depletion of CD8+
lymphocytes but not by depletion of CD4+ lymphocytes (Fig. 5A). The different cytokine effect on the HBV DNA
vaccine-induced CTL activity was further confirmed by assays using
another HBsAg-transfected target cells, CT26/S, which produced more
HBsAg than P815/S and was found to be more sensitive to the CTL assay.
As shown in Figure 5B, HBsAg-specific cytolysis was enhanced
by coinjection of the IL-12 gene but suppressed by the IL-4 gene (Fig. 5B). This cytolytic activity was blocked by an
anti-CD8 mAb but not by an anti-CD4 mAb (Fig. 5B). Taken together, these results indicate that the
CTL activity primed by a HBV DNA vaccine in vivo is mediated by cells
expressing CD4-CD8+ surface phenotype and the
CTL activity can be enhanced or suppressed depending on the cytokine
gene coexpressed.
|
To determine the effect of phenotype changes of immune responses
on protective efficacy in vivo, the ability of the pS DNA vaccine in
combination with IL-12 or IL-4 gene to immunoprotect against
transplantation of syngeneic HBsAg-transfected tumor cells was
examined. Groups of three to five mice were vaccinated once with 100
µg of pS plus 100 µg of pcDNA3 or vectors encoding IL-4 or IL-12.
Mice that received pcDNA3 alone served as a negative control group.
Three weeks after vaccination, all mice were inoculated with 1 x
105 HBsAg-expressing CT26/S or the parental CT26 tumor
cells. All mice that received pcDNA3 and hence were not immune to HBsAg
developed rapidly growing CT26 or CT26/S tumors within 2 wk after
transplantation (Fig. 6, A and
E). Four of five mice immunized with pS + pcDNA3
and challenged with CT26/S showed an inhibition of tumor growth (Fig. 6B). The protective efficacy was dramatically
increased when the IL-12 gene was coinjected with plasmid pS; tumor
growth was significantly suppressed, and two of five mice remained
tumor free up to 60 days following tumor challenge (Fig. 6D). The tumor immunity is HBsAg specific, since mice
immunized with pS + pcDNA3 (Fig. 6F) or pS
+ pIL-12 (Fig. 6H) and challenged with the parental
CT26 cells showed little protection. In contrast, vaccination with
pS + pIL-4 offered little if any protection against CT26/S (Fig. 6C) or CT26 (Fig. 6G) challenge.
These results demonstrate that the difference in phenotype of immune
responses stimulated by DNA vaccination under influence of various
cytokines has a significant effect on protective efficacy against tumor
challenge.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although the genetic vaccination approach has been applied to the development of DNA vaccines against many different pathogens, the efficacy of different DNA vaccines has varied widely. Some DNA vaccines are incapable of inducing specific immune responses even after several inoculations of large amounts of plasmid DNA (7, 43, 44). Thus, improvement of vaccine efficacy has become a critical issue for the acceptance of DNA vaccines as a standard vaccination technology. One explanation for the suboptimal immune responses induced by some DNA vaccines may be related to the cellular location of the encoded Ag. Indeed, it was found that the effectiveness of genetic immunization with a minigene coding for single epitopes was significantly enhanced if the epitope sequence was fused in frame with the adenovirus E3 leader sequence to target the epitope to the endoplasmic reticulum (59). Targeting of viral Ag for rapid cytoplasmic degradation was also reported to enhance de novo CTL responses in vivo (60). In contrast, it was reported that plasmids encoding a cytoplasmic/membrane or a secreted form of Ag have little effect on the ability of the plasmids to elicit humoral and cellular immune responses (33, 61, 62). Other approaches to enhance the efficacy of DNA vaccines include fusion of the encoded Ag with a stronger immunogen as a carrier (11), coexpression with B7-1/B7-2 accessory molecules (63, 64), or immunization with plasmid DNA-transfected dendritic cells (65).
Our laboratory has sought to promote and modulate immune responses to a
DNA vaccine through codelivery of various cytokine genes. The rationale
is based on our previous findings that a weak tumor Ag can be converted
into a strong immunogen when it is conjugated to GM-CSF (39), IL-2, or
IL-4 (38). It has also been reported that cytokines such as IL-1ß,
IL-2, or IFN- were able to enhance specific immune responses when
administered along with the Ag for prolonged periods (66, 67). More
direct evidence that cytokines can influence the efficacy of DNA
vaccination was shown by Irvine et al. (68). These authors demonstrated
that, in a mouse tumor model, when recombinant IL-2, IL-6, IL-7, or
IL-12 was added following administration of DNA encoding a
tumor-associated Ag, the number of metastases was significantly reduced
compared with that in mice treated with DNA only. To avoid the
potential side effects associated with systemic administration of
recombinant cytokines, we reason that direct injection of plasmids
containing an Ag and a cytokine gene may be able to achieve a sustained
but low level of cytokines, which are delivered to tissues in which
immune interactions take place. Using this approach, we have shown
previously that plasmids coexpressing IL-2 and HBsAg in the fusion or
nonfusion context resulted in at least a 100-fold increase in the
ability of the plasmids to induce Ab and T cell proliferative responses
to HBsAg (33). In the present study, we attempt to accomplish local
concentrations of cytokines by another strategy, that is, coinjection
of cytokine genes with the HBV DNA vaccine. It was found that
coinjection of the GM-CSF, IL-2, IL-12, or IFN- gene enhanced T cell
proliferation by two- to threefold, while coadministration of the IL-4
gene achieved only a slight increase in this response (Table I
and Fig. 1). Ab responses to the DNA vaccine were also affected by coexpression
of cytokines. While GM-CSF, IL-2, IL-4, and IL-12 increased both IgM
and IgG anti-HBs titers, IFN- was unique in that it increased
IgM but suppressed IgG Ab (Fig. 2). We found that the adjuvant effect
of the cytokine-expressing vectors was dependent upon coinjection with
the plasmid encoding HBsAg; inoculation of the two plasmids separately
had no effect on the magnitude of specific immune responses, indicating
that colocalization of cytokines and Ags at the site of immune
interaction was important for the observed cytokine adjuvant effect. It
is noteworthy that the cytokine adjuvant effect is most significant
within the first 2 to 5 mo following DNA vaccination and becomes
negligible thereafter (data not shown), suggesting that the transfected
muscle cells may be eliminated by that time. The enhancing effect of
cytokine genes on immune responses to DNA vaccines has also been
reported in other studies. In a rabies virus model, coinjection of
plasmids encoding GM-CSF but not IFN- enhanced immune responses to
the DNA vaccine (69). Similarly, plasmids encoding an idiotypic Ag of B
cell lymphoma with its carboxyl-terminal end conjugated to GM-CSF or a
nine-amino acid peptide derived from IL-1ß were shown to induce
enhanced Ab titers and to provide protective immunity against a
subsequent lethal tumor challenge (19, 20). It has been reported
recently that plasmid vectors containing an unmethylated CpG
dinucleotide motif can elicit much stronger humoral and cellular
responses to the encoded Ag than vectors that do not contain this
sequence (42). The adjuvant effect of the cytokine vectors could be
attributed to the presence of the CpG motif in the coding sequences of
cytokines instead of functioning through their biologic effects. Our
studies provide evidence that rules out this possibility, since
coinjection of the control plasmid pcDNA3, which was used to construct
the cytokine vectors, did not increase immune responses to the HBV DNA
vaccine. Furthermore, none of the three potential immune-enhancing CpG
motifs, i.e., GACGTC, AGCGCT, and AACGTT, was present in the coding
sequence of murine IL-2, IL-4, GM-CSF, IFN-, or the IL-12 p40 genes.
Among those tested, IL-12 p35 is the only gene containing one such
motif; however, it is unlikely that this CpG motif by itself could make
the immune phenotype changes observed in the IL-12 gene-injected
mice.
Development of the appropriate CD4+ Th cell subset
during an immune response is critical for eradication of infectious
organisms. A functional consequence of Th1 activation is the promotion
of cell-mediated immune responses characterized by expression of
C-fixing and opsonizing Abs, such as IgG2a in the mouse, as well as
macrophage and CTL activation (28, 29). In contrast, Th2 cells promote
development of selected humoral immune responses, including expression
of IgG1 and IgE (28, 29). The mechanisms directing the development of
naive CD4 cells into Th1 or Th2 effectors include the presence of
certain cytokines at the site of primary Ag stimulation of naive cells,
the effective concentration of Ag presented to T cells, and the nature
of APC (70). Among these factors, the presence of local cytokines plays
the most critical role in shaping the nature of immune responses. IL-4
is essential for priming Th2 cells (30), and IL-12 and IFN- strongly
support the development of Th1 cells (31, 32). Most of the above
conclusions were obtained from in vitro studies of naive Th cells
stimulated with mitogen or Ag in the presence of various cytokines or
from in vivo studies in which the exogenous cytokines were applied
systematically. Codelivery of cytokine genes with the DNA vaccines can
provide a sustained but low level of cytokines to tissues of immune
interactions and may thus represent a more appropriate model for
gaining insight into the differentiation of naive Th cells during an
immune response. Our studies confirm the importance of IL-12 and
IFN- for the development of Th1 cells and IL-4 for Th2 cells.
Immunization of the HBV DNA vaccine with plasmids encoding IL-12 or
IFN- produced T cells that make significant amounts of IL-2 and
IFN- but no IL-4 in response to HBsAg (Fig. 3), indicating a shift
toward Th1 immunity. This observation is corroborated by the in vivo
increase of HBsAg-specific IgG2a Ab (Fig. 4B) and
enhancement of CTL activity (Fig. 5, A and
B). In contrast, coexpression of IL-4 induced a shift
toward the production of Th2 cytokine (Fig. 3C),
which is further confirmed by the in vivo increase of specific IgG1
production (Fig. 4A) and decrease of IgG2a (Fig. 4B)
and CTL activity (Fig. 5, A and B).
Coexpression of IL-2 or GM-CSF with the HBV DNA vaccine favored the
generation of Th1 cells (Fig. 3, A and B);
however, the Th2 cell response was less affected (Fig. 3C). This effect was accompanied by an enhanced
production of both IgG1 (Fig. 4A) and IgG2a (Fig. 4B) isotypes as well as by CTL activity (Fig. 5A). We found that there was a strong correlation of
CTL activity in immunized animals with protective efficacy against
syngeneic HBsAg-transfected tumor cells. Mice that received a mixture
of the HBV DNA vaccine and the control vector showed an inhibition of
tumor growth (Fig. 6B). The protective efficacy was
dramatically increased when the DNA vaccine was coinjected with the
IL-12 gene, which helps elicit the strongest CTL activity among various
cytokine genes tested. In contrast, vaccination with the DNA vaccine in
the presence of the IL-4 gene, which favored Th2 response and
suppressed CTL activity, abolished the protective immunity (Fig. 6C). These results indicate that, by codelivering
DNAs encoding Ag and specific cytokines, it is possible to regulate the
quantitative and qualitative nature of an immune response to make it
more effective in dealing with pathogens. We also found that the in
vitro CTL activity of the immunized splenocytes is specific for HBsAg
and can be removed by depletion of CD8+ cells (Fig. 5A) or by addition of an anti-CD8 mAb (Fig. 5B) in the culture medium, but not by depletion of
CD4+ cells or by the CD4-specific mAb, indicating that the
CTL activity is mediated by CD8+ lymphocytes. It was
demonstrated that the CTL generated by HBsAg DNA immunization is
restricted by class I MHC and the Ld-restricted S28–39
epitope is the only CTL epitope of HBsAg in H-2d BALB/c
mice (71). Similar findings on the regulation of Th1 vs Th2 populations
by the IL-2, IL-4, or GM-CSF gene in the immune response to a hepatitis
C virus DNA vaccine has also been reported previously (72). In another
study, Kim et al. have shown that codelivery of the IL-12 gene along
with DNA vaccine formulations for HIV-1 Ag resulted in the reduction of
specific Ab response, whereas the T cell-proliferative reaction and CTL
activity were increased (73). It is noteworthy that coexpression of the
IL-12 gene with the HIV-1 DNA vaccine caused splenomegaly and increased
the number of white blood cells in the immunized spleen, a phenomenon
also reported by in vivo administration of rIL-12 (74, 75, 76). In our
study, we found that codelivery of the IL-12 gene, as well as the other
cytokine genes tested, did not lead to enlargement of the spleen (data
not shown) or changes of frequency of CD4+ and
CD8+ cells in the spleen. What caused the discrepancy
between these two studies is not clear, but it may be due to the amount
of IL-12 produced by the different IL-12 vectors.
HBV is one of several viruses that can persist after primary infection
in humans, subsequently causing chronic necroinflammatory liver disease
and hepatocellular carcinoma. The currently used therapeutic approaches
to eliminating the virus and terminating chronic infections have not
been very successful. It is widely acknowledged that, in humans, T
cells play a critical role in clearing HBV infections and in inducing
liver lesions associated with persistent HBV infections (77).
Therefore, the idea of vaccinating HBV chronic carriers to control
viral infections is challenged mainly due to a concern of possible
massive liver damage mediated by immune cells. However, a recent report
has shown that adoptively transferred HBsAg-specific CTL can abolish
HBV gene expression and replication in HBV transgenic mice by secreting
IFN- and TNF- (78). Most importantly, these cytokines perform
their antiviral activity mainly by a noncytopathic process. These
results raise the possibility that chronic HBV infection may be
controlled by induction of appropriate T cell subsets, which can
produce the therapeutic cytokines. Indeed, using a similar transgenic
model, it was reported that T cell-mediated immunity induced by a HBV
DNA vaccine resulted in the complete clearance of circulating HBsAg and
in the long-term control of transgene expression in hepatocytes (79).
Again, no detectable cytopathic effect was found to accompany this
therapeutic response. HBV DNA vaccine has also been shown to induce
high titers of anti-HBs Ab in chimpanzees (80), indicating its
potential application in human trials. According to the studies and
data presented here, we believe that the use of DNA vaccines in
combination with an appropriate cytokine gene to regulate the phenotype
of immune responses may provide an effective treatment for chronic HBV
infections.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Mi-Hua Tao, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529. E-mail address:
3 Abbreviations used in this paper: HBV, hepatitis B virus; HBsAg, hepatitis B virus surface Ag; GM-CSF, granulocyte-macrophage colony-stimulating factor.
Received for publication January 23, 1997. Accepted for publication October 20, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and diminishes IL-4 inhibition in such priming. Proc. Natl. Acad. Sci. USA 90:10188.ß-TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-g production is IFN-g-dependent. Int. Immunol. 5:1119.This article has been cited by other articles:
|
|
 |
|
  E. Zeira, A. Manevitch, Z. Manevitch, E. Kedar, M. Gropp, N. Daudi, R. Barsuk, M. Harati, H. Yotvat, P. J. Troilo, et al. Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization FASEB J, November 1, 2007; 21(13): 3522 - 3533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hu, X. Lu, L. Tao, B. Bai, Z. Zhang, Y. Chen, F. Zheng, J. Chen, Z. Chen, and H. Wang Induction of Specific Immune Responses by Severe Acute Respiratory Syndrome Coronavirus Spike DNA Vaccine with or without Interleukin-2 Immunization Using Different Vaccination Routes in Mice Clin. Vaccine Immunol., July 1, 2007; 14(7): 894 - 901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wang, S. A. Gujar, L. Cova, and T. I. Michalak Bicistronic Woodchuck Hepatitis Virus Core and Gamma Interferon DNA Vaccine Can Protect from Hepatitis but Does Not Elicit Sterilizing Antiviral Immunity J. Virol., January 15, 2007; 81(2): 903 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Schott Immunesurveillance by dendritic cells: potential implication for immunotherapy of endocrine cancers. Endocr. Relat. Cancer, September 1, 2006; 13(3): 779 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Li, X. Yang, Y. Jiang, and J. Liu A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice Int. Immunol., October 1, 2005; 17(10): 1293 - 1302. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Mwangi, W. C. Brown, G. A. Splitter, Y. Zhuang, K. Kegerreis, and G. H. Palmer Enhancement of antigen acquisition by dendritic cells and MHC class II-restricted epitope presentation to CD4+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection J. Leukoc. Biol., August 1, 2005; 78(2): 401 - 411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Teramoto, K. Kontani, Y. Ozaki, S. Sawai, N. Tezuka, T. Nagata, S. Fujino, Y. Itoh, O. Taguchi, Y. Koide, et al. Deoxyribonucleic Acid (DNA) Encoding a Pan-Major Histocompatibility Complex Class II Peptide Analogue Augmented Antigen-specific Cellular Immunity and Suppressive Effects on Tumor Growth Elicited by DNA Vaccine Immunotherapy Cancer Res., November 15, 2003; 63(22): 7920 - 7925. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Lo, S.-C. Lee, P.-Y. Wu, W.-Y. Pan, J. Su, C.-W. Cheng, S. R. Roffler, B.-L. Chiang, C.-N. Lee, C.-W. Wu, et al. Antitumor and Antimetastatic Activity of IL-23 J. Immunol., July 15, 2003; 171(2): 600 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Osorio and H. Ghiasi Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing TH1 and TH2 Cytokine Genes J. Virol., May 15, 2003; 77(10): 5774 - 5783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Z. Wu, J.-P. Zhao, Y. Wan, Z.-C. Jia, W. Zhou, J. Bian, B. Ni, L.-Y. Zou, and Y. Tang Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo J. Virol., September 11, 2002; 76(20): 10264 - 10269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. A. van Rooij, H. L. Glansbeek, L. A. T. Hilgers, E. G. te Lintelo, Y. E. de Visser, W. J. A. Boersma, B. L. Haagmans, and A. T. J. Bianchi Protective Antiviral Immune Responses to Pseudorabies Virus Induced by DNA Vaccination Using Dimethyldioctadecylammonium Bromide as an Adjuvant J. Virol., September 11, 2002; 76(20): 10540 - 10545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A Poland, D. Murray, and R. Bonilla-Guerrero Science, medicine, and the future: New vaccine development BMJ, June 1, 2002; 324(7349): 1315 - 1319. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Haupt, M. Roggendorf, and K. Mann The Potential of DNA Vaccination against Tumor-Associated Antigens for Antitumor Therapy Experimental Biology and Medicine, April 1, 2002; 227(4): 227 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. L. Glansbeek, B. L. Haagmans, E. G. te Lintelo, H. F. Egberink, V. Duquesne, A. Aubert, M. C. Horzinek, and P. J. M. Rottier Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus J. Gen. Virol., January 1, 2002; 83(1): 1 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Moore, W.-p. Kong, B. K. Chakrabarti, and G. J. Nabel Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice J. Virol., January 1, 2002; 76(1): 243 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V Schmitz, C Qian, J Ruiz, B Sangro, I Melero, G Mazzolini, I Narvaiza, and J Prieto Gene therapy for liver diseases: recent strategies for treatment of viral hepatitis and liver malignancies Gut, January 1, 2002; 50(1): 130 - 135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Planelles, M. C. Thomas, C. Alonso, and M. C. Lopez DNA Immunization with Trypanosoma cruzi HSP70 Fused to the KMP11 Protein Elicits a Cytotoxic and Humoral Immune Response against the Antigen and Leads to Protection Infect. Immun., October 1, 2001; 69(10): 6558 - 6563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hanlon, D. Argyle, D. Bain, L. Nicolson, S. Dunham, M. C. Golder, M. McDonald, C. McGillivray, O. Jarrett, J. C. Neil, et al. Feline Leukemia Virus DNA Vaccine Efficacy Is Enhanced by Coadministration with Interleukin-12 (IL-12) and IL-18 Expression Vectors J. Virol., September 15, 2001; 75(18): 8424 - 8433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Bosarge, J. M. Watt, D. O. McDaniel, E. Swiatlo, and L. S. McDaniel Genetic Immunization with the Region Encoding the {alpha}-Helical Domain of PspA Elicits Protective Immunity against Streptococcus pneumoniae Infect. Immun., September 1, 2001; 69(9): 5456 - 5463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
F. Siegel, M. Lu, and M. Roggendorf Coadministration of Gamma Interferon with DNA Vaccine Expressing Woodchuck Hepatitis Virus (WHV) Core Antigen Enhances the Specific Immune Response and Protects against WHV Infection J. Virol., June 1, 2001; 75(11): 5036 - 5042. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. Haupt, F. Siegel, M. Lu, D. Yang, G. Hilken, K. Mann, M. Roggendorf, and B. Saller Induction of a Cellular and Humoral Immune Response against Preprocalcitonin by Genetic Immunization: A Potential New Treatment for Medullary Thyroid Carcinoma Endocrinology, March 1, 2001; 142(3): 1017 - 1023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-I. Sin, J. Kim, C. Patchuk, and D. B. Weiner Interleukin 7 Can Enhance Antigen-Specific Cytotoxic-T-Lymphocyte and/or Th2-Type Immune Responses In Vivo Clin. Vaccine Immunol., September 1, 2000; 7(5): 751 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Chen, T.-L. Wang, C.-F. Hung, Y. Yang, R. A. Young, D. M. Pardoll, and T-C. Wu Enhancement of DNA Vaccine Potency by Linkage of Antigen Gene to an HSP70 Gene Cancer Res., February 1, 2000; 60(4): 1035 - 1042. [Abstract] [Full Text]
|
|
|
|
|
|
H.-W. Chen, C.-H. Pan, M.-Y. Liau, R. Jou, C.-J. Tsai, H.-J. Wu, Y.-L. Lin, and M.-H. Tao Screening of Protective Antigens of Japanese Encephalitis Virus by DNA Immunization: a Comparative Study with Conventional Viral Vaccines J. Virol., December 1, 1999; 73(12): 10137 - 10145. [Abstract] [Full Text]
|
|
|
|
|
|
C. Jiang, D. M. Magee, and R. A. Cox Coadministration of Interleukin 12 Expression Vector with Antigen 2 cDNA Enhances Induction of Protective Immunity against Coccidioides immitis Infect. Immun., November 1, 1999; 67(11): 5848 - 5853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Tam, C. Lim, J. Bard, and B. Pai Contact Hypersensitivity Responses Following Ribavirin Treatment In Vivo Are Influenced by Type 1 Cytokine Polarization, Regulation of IL-10 Expression, and Costimulatory Signaling J. Immunol., October 1, 1999; 163(7): 3709 - 3717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Rodrigues, M. Ribeirao, V. Pereira-Chioccola, L. Renia, and F. Costa Predominance of CD4 Th1 and CD8 Tc1 Cells Revealed by Characterization of the Cellular Immune Response Generated by Immunization with a DNA Vaccine Containing a Trypanosoma cruzi Gene Infect. Immun., August 1, 1999; 67(8): 3855 - 3863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Jiang, D. M. Magee, and R. A. Cox Construction of a Single-Chain Interleukin-12-Expressing Retroviral Vector and Its Application in Cytokine Gene Therapy against Experimental Coccidioidomycosis Infect. Immun., June 1, 1999; 67(6): 2996 - 3001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Adam. D. Cohen, J. D. Boyer, and D. B. Weiner Modulating the immune response to genetic immunization FASEB J, December 1, 1998; 12(15): 1611 - 1626. [Abstract] [Full Text]
|
|
|
|
|
|
B. Wizel, N. Garg, and R. L. Tarleton Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection Infect. Immun., November 1, 1998; 66(11): 5073 - 5081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chun, M. Daheshia, N. A. Kuklin, and B. T. Rouse Modulation of Viral Immunoinflammatory Responses with Cytokine DNA Administered by Different Routes J. Virol., July 1, 1998; 72(7): 5545 - 5551. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
