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Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
Wyeth Lederlie Vaccines, Malvern, PA 19355
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and chemokines (RANTES and macrophage
inflammatory protein-1
) were significantly increased by IL-12
coinjection. However, the production of cytokines (IL-4 and IL-10) and
chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection
with a gD DNA vaccine showed significantly better protection from
lethal HSV-2 challenge compared with gD DNA vaccination alone in both
inbred and outbred mice. This enhanced protection appears to be
mediated by CD4+ T cells, as determined by in vivo
CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine
adjuvant drives Ag-specific Th1 type CD4+ T cell responses
that result in reduced HSV-2-derived morbidity as well as
mortality. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK
cells 2 . Several studies have reported that IL-12 as a protein has a critical role in inducing antiviral and antitumor effects in vivo. Direct administration of IL-12 protein or cDNA expressing IL-12 by gene gun can affect tumor progression and metastasis in animal models 4, 5, 6 . Similarly, therapeutic treatments with IL-12 protein can result in protective responses to some infectious viral agents, including vesicular stomatitis virus 7 , CMV 8 , and lymphocytic choriomeningitis virus 9 . Induction of IL-12 has also been related to viral clearance in chronic hepatitis B virus in humans 10 . Most recently, it has been reported that administration of IL-12 protein has some potential to treat lethal herpes simplex virus (HSV)3 infection in mice 11 . Furthermore, protective adjuvant effects of IL-12 protein coadministration have been observed in mouse bacterial infection models 12 . When used as a molecular adjuvant, IL-12 cDNA induces Ag-specific CTL responses with inhibitory effects on humoral responses in HIV DNA vaccine studies 13 . Iwasaki et al. 14 similarly reported that IL-12 cDNA codelivery with DNA immunogen encoding for influenza NP resulted in enhanced cellular immune responses.
HSV-specific humoral and cellular immune responses have been suggested to be responsible for protective immunity against HSV infection. Recurrence of latent HSV has been linked to decreased cellular responses in the guinea pig model 15 . It has also been reported that adoptive transfer of HSV-specific T cells results in protecting animals from lethal HSV challenges 16 . Moreover, HSV-specific Th1 type CD4+ T cells have been reported to be responsible for protective immunity against intravaginal HSV challenge 17 . On the other hand, HSV-specific Abs, which are highly present in humans and mice, are insufficient to prevent central nervous system infection from HSV 18 . This suggests that cell-mediated immunity is directly related to inhibition of HSV replication and its spread to other tissues. Enhancement of therapeutic immune responses by cytokine gene delivery is an important goal of gene therapy for infectious diseases. Furthermore, there have been no HSV studies on the effect of DNA vaccines enhanced by IL-12 gene delivery and no infectious challenge studies.
In this study we observed that injection of IL-12 cDNA can activate resting immune cells. When coinjected with a DNA vaccine, IL-12 cDNA drives protective immune responses to Ag-specific Th1-type CD4+ T cell-mediated types while inhibiting the generation of Ab responses. Lastly, IL-12 dosage studies in a herpes mouse model indicate that the dose ratio of DNA vaccine to adjuvants is a factor to be considered for induction of immune responses to and protection from viral infection. These results support the idea that IL-12 plasmid cytokine gene delivery can modulate immune responses to infectious agents in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female inbred BALB/c mice (4–6 wk) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Female outbred Swiss-Webster mice (6–7 wk) were purchased from Taconic Farms (Germantown, NY). Their care was performed under the guidelines of the National Institutes of Health (Bethesda, MD) and Institutional Animal Care and Use Committee-approved protocols.
Reagents
HSV-2 strain 186 (a gift from Dr. P. Schaffer, University of Pennsylvania, Philadelphia, PA) and HSV-2 strain 333 were propagated in the Vero cell line (American Type Culture Collection, Manassas, VA). DNA vaccine pgD (pAPL-gD2) encoding HSV-2 gD protein was previously described 19 . The expression vectors, pCDNA3-IL-12 p35, pCDNA3-IL-12 p40, and pCDNA3-GM-CSF were previously constructed in our laboratory. The procedures for their construction and expression have been previously described 13 . Plasmid DNA was produced in bacteria and was purified by double-banded CsCl preparations. Recombinant HSV-2 gD proteins, a gift from Drs. G. Cohen and R. Eisenberg (University of Pennsylvania), were used as recombinant Ag in these studies.
DNA inoculation of mice
The quadriceps muscles of BALB/c mice were injected with pgD constructs formulated in 100 µl of PBS and 0.25% bupivacaine-HCl (Sigma, St. Louis, MO) using a 28-gauge needle (Becton Dickinson, Franklin Lakes, NJ). The pCDNA3-IL-12 p35 and pCDNA-IL-12 p40 expression cassettes were mixed with pgD plasmid solution before injection.
ELISA
HSV-2 gD protein (0.75 µg/ml in PBS) was adsorbed onto 96-well microtiter plates. After blocking the plates for 1 h with blocking buffer (PBS, 0.05% Tween-X, and 2% BSA), 50 µl of diluted sera (1/100 in PBS) were added, and the plates were incubated at 37°C for 1 h. After addition of anti-murine IgG conjugated with horseradish peroxidase (HRP), the plates were incubated for 1 h at 37°C. For the determination of vaginal IgA levels, the vaginal route of each animal was washed with 200 µl of PBS, and then 50 µl of the washed solution was reacted with gD-2 protein in ELISA. For the determination of IgA levels, anti-murine IgA-HRP (Zymed, South San Francisco, CA) was substituted for anti-murine IgG-HRP (Sigma). For the determination of IgE levels, rat IgG (Zymed) raised against murine IgE and anti-rat IgG-HRP (Sigma) were substituted for anti-murine IgG-HRP. This was followed by addition of the ABTS (2,2'-azino-bis[3-ethylbenzenzthiazoline-6-sulfonic acid]) substrate solution (Chemicon, Temecula, CA). In each step, plates were washed three times with the wash buffer (PBS and 0.05% Tween-X). The plates were read on a Dynatech MR5000 plate reader with the OD at 405 nm. The ELISA titers were determined as the reverse of the highest serum dilution showing the same OD value as sera of naive mice.
Th cell proliferation assay
Spleens were aseptically removed from each group and pooled. Lymphocytes were harvested from spleens and prepared as the effector cells by removing the erythrocytes and by washing several times with fresh medium (RPMI 1640 supplemented with 10% FCS) as previously described 13 . The isolated cell suspensions were resuspended to a concentration of 1 x 106 cells/ml. A 100-µl aliquot containing 1 x 105 cells was immediately added to each well of a 96-well microtiter flat-bottom plate. HSV-2 gD protein at a final concentration of 1 and 5 µg/ml was added to wells in triplicate. The cells were incubated at 37°C in 5% CO2 for 3 days. One microcurie of tritiated thymidine was added to each well, and the cells were incubated for 12–18 h at 37°C. The plate was harvested, and the amount of incorporated tritiated thymidine was measured in a beta plate reader (Wallac, Turku, Finland). The stimulation index was determined from the formula: stimulation index = (experimental count - spontaneous count)/spontaneous count. Spontaneous count wells included 10% FCS, which served as an irrelevant protein control. To assure that cells were healthy, 5 µg/ml PHA (Sigma) was used as a polyclonal stimulator positive control.
Th1 and Th2 type cytokines and chemokines
A 1-ml aliquot containing 6 x 106 splenocytes
was added to wells of 24-well plates. Then, 1 µg of HSV-2 gD
protein/ml was added to each well. After 2-day incubation at 37°C in
5% CO2, cell supernatants were secured and then used for
detecting levels of IL-2, IL-4, IL-10, IFN-, RANTES, MCP-1, and
MIP-1 using commercial cytokine kits (BioSource (Camarillo, CA) and
R & D Systems (Minneapolis, MN) by adding the extracellular fluids to
the cytokine- or chemokine-specific ELISA plates.
FACS analysis
Spleen cells (1 x 105) were washed three times with FACS buffer (PBS, 1% BSA, and 0.1% sodium azide) and then reacted with phycoerythrin-conjugated anti-mouse Ly-6A/E (PharMingen, San Diego, CA) for 30 min on ice. After washing three times with FACS buffer, cells were analyzed for the percentage of Ly6-positive cells on a flow cytometer (EPICS XL, Coulter, Hialeah, FL).
In vivo deletion of CD4+ T cells
One hundred microliters of anti-CD4 (clone GK1.5) ascites fluid (a gift from Dr. N. Chirmule of University of Pennsylvania) was administered i.p. on days -3, 0, and 3 of viral challenge. Ab treatment resulted in >98% depletion of specific CD4+ T cell subsets of representative animals over a 3-wk period. Depleted mice were subsequently challenged with virus on day 0.
Intravaginal HSV-2 challenge
Three or five weeks after the last DNA injection, inbred BALB/c mice were challenged intravaginally with 4 x LD50 of HSV-2 strain 186 (1.4 x 104 plaque-forming units). Before inoculating the virus, the intravaginal area was swabbed with a cotton-tipped applicator (Hardwood Products, Guiford, ME) soaked with 0.1 M NaOH solution and then cleaned with dried cotton applicators. In the case of outbred Swiss-Webster mice, animals were challenged intravaginally with 3 x LD50 of HSV-2 strain 333 (5 x 105 plaque-forming units) after 21 days of DNA injection as described previously 20 . Mice were then examined daily to evaluate pathological conditions and survival rates.
Statistical analysis
Statistical analysis was performed using paired Student’s t test and analysis of variance. Values between different immunization groups were compared. A p value <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice have a few Ly6 resting peripheral lymphocytes, and
IL-12 activation of immune cells leads to expression of Ly6 Ag 21, 22 . Therefore, Ly6 expression can be used as a marker of in vivo
delivery of IL-12 genes. To determine whether IL-12 levels driven by
our DNA vaccine cassettes can influence immune cell activation in vivo,
expression levels of Ly6 on the splenocytes were detected using FACS
analysis. Fig. 1
shows that injection of
50 µg of IL-12 genes significantly increased the percentage of
splenocytes expressing Ly6 to 24% on day 3. Ly6 expression levels
peaked (32%) on day 6 and then continued to decline over the time
period studied. By 60 days there was no difference between DNA-injected
and negative control animals (7%). In contrast, injection of 0.1 µg
of IL-12 genes had little effect on Ly6 expression on splenocytes.
Therefore, the in vivo plasmid delivery of IL-12 can have a significant
effect on cell activation status in vivo.
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To determine whether coinjection of gD genetic vaccines with IL-12
expression vectors might influence humoral immune responses against gD,
sera and vaginal fluids obtained 2 wk after each DNA inoculation were
tested in ELISA. As shown in Fig. 2, gD
DNA vaccine induced systemic gD-specific IgG, IgE, and IgA levels
significantly higher than negative control values, whereas coinjection
with IL-12 genes inhibited systemic gD-specific IgG, IgE, and IgA
production significantly more than gD DNA vaccine. We also investigated
inhibitory effects of IL-12 coinjection on Ab production over the time
period (Fig. 3). As shown in Fig. 3A, gD DNA vaccine induced systemic gD-specific IgG
levels significantly higher than negative control values, whereas
coinjection with IL-12 significantly decreased the overall IgG levels.
This inhibition of IgG production was more significant after an 8 wk
booster injection with pgD and IL-12 genes. Since HSV-2 infects its
host mucocutaneously, it is also important to measure levels of IgA in
vaginal fluid. Fig. 3B shows that gD DNA vaccine induced
gD-specific intravaginal IgA levels. However, coinjection with IL-12
decreased the local anti-gD IgA levels, similar to the decrease
observed in serum IgG levels. However, GM-CSF cDNA coinjection, used as
a parallel positive control, enhanced systemic IgG and local IgA
production significantly higher than gD DNA vaccine alone (data not
shown).
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Th cells play an important role in eliciting both humoral and
cellular immune responses via expansion of Ag-stimulated B cells and
expansion of CD8+ T cells, respectively. As a specific
indicator of CD4 activation, T cell proliferation was examined. The
gD-2 protein (1 and 5 µg/ml) was used for Ag-specific stimulation of
T cells. For a positive control, 5 µg/ml PHA was used as a polyclonal
stimulator. As shown in Fig. 4, a low
background level of Th cell proliferation was observed in negative
controls. However, gD DNA vaccine-stimulated cells had an enhanced Th
cell proliferative response. When coinjected with IL-12 genes, Th cell
proliferation levels were further increased compared with the effect of
gD DNA vaccine alone.
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Th1 cytokines (IL-2 and IFN-) and Th2 cytokines (IL-4, IL-5,
and IL-10) are major parameters in our understanding of the
polarization of immune responses. Th1 immune responses are thought to
drive induction of cellular immunity, whereas Th2 immune responses
preferentially drive humoral immunity. We examined the effects of
coinjection of gD DNA vaccine with and without IL-12 genes on Th1 or
Th2 phenotypic change. As shown in Fig. 5, IL-2, IL-10, and IFN- production
was increased by the gD DNA vaccine compared with negative control
values. In contrast, IL-4 levels were unaffected by the gD DNA vaccine.
When coinjected with IL-12 genes, IL-2 and IFN- production was
further boosted. In contrast, IL-4 and IL-10 production was decreased
by IL-12 coinjection.
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ß chemokines (C-C type), including RANTES, MIP-1, and MCP-1
chemoattract particularly monocytic phagocytes and activate T cells,
basophils, eosinophils, and mononuclear phagocyte as well as a variety
of other soluble immune modulators 23, 24 . Compared with MCP-1,
RANTES and MIP-1 were also reported to be major HIV-suppressive
factors 25 . These molecules are thought to be important in modulating
inflammatory immune responses. However, their direct role in infectious
diseases is under investigation. The relationship of IL-12 as a vaccine
adjuvant to chemokine production in vivo is unknown. We investigated
the levels of chemokines (RANTES, MCP-1, and MIP-1) induced by IL-12
coinjection. As shown in Fig. 6, gD DNA
vaccine alone enhanced the production of RANTES, MCP-1, and MIP-1 in
an Ag-specific manner. Coinjection with IL-12 cDNA enhanced RANTES and
MIP-1 production significantly higher than gD DNA vaccine alone. In
contrast, MCP-1 production was decreased by IL-12 coinjection. This
modulation supports the idea that IL-12 can have specific effects on
individual members of the ß chemokine family
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Inbred mice (BALB/c) surviving after challenge with 4 x
LD50 of HSV-2 (186) were followed for 1 mo postchallenge to
examine pathological symptoms. Herpetic lesions were observed on the
epithelial layers of the skin around the vaginal area. As shown in
Table I, immunization with gD DNA vaccine
alone resulted in more mice showing herpetic lesions than those that
received the pgD and IL-12 immunization. Furthermore, coinjection with
IL-12 genes resulted in complete healing of lesions in all surviving
animals 19 days after viral challenge. Animals vaccinated with gD DNA
vaccine alone failed to recover from their lesions even 21 days after
viral challenge. The herpetic lesions were scored based upon their
severity. As shown in Fig. 7A,
coinjection with IL-12 genes induced a significantly lower degree of
severity in herpetic lesions compared with gD DNA vaccine alone.
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, immunization with gD DNA vaccine alone reduced the number
of mice showing herpetic lesions compared with the effect of negative
plasmid control or IL-12 cDNA alone. However, coinjection with IL-12
cDNA significantly reduced the number of mice with lesions over the
time period. Fig. 7B shows that injection of IL-12 genes
alone reduced morbidity scores slightly lower than negative plasmid
controls. When immunized with gD DNA vaccine alone, the morbidity score
was significantly reduced over the time period studied compared with
that in negative control animals or IL-12-injected animals. When
coinjected with pgD plus IL-12 genes, the morbidity score was the most
significantly decreased. Effects of IL-12 doses on Ab responses
To determine whether coinjection with a different dose of IL-12
genes influences systemic IgG levels in vivo, mice were coimmunized
with IL-12 genes ranging from 5–40 µg/mouse, and then sera obtained
4 wk after DNA inoculation were tested in ELISA. As shown in Fig. 8, gD-specific IgG levels were induced
significantly by coinjection with 5 µg of IL-12 genes. However, when
coinjected with 20 or 40 µg of IL-12 genes, gD-specific IgG levels
were completely inhibited to levels of negative controls. ELISA titers
of equally pooled sera of pgD alone (6 µg), pgD plus IL-12 (5 µg),
pgD plus IL-12 (20 µg), and pgD plus IL-12 (40 µg) were also
determined to be 160, 320, 40, and 40, respectively. The pCDNA3 has
been routinely used as a negative control and had no effect on ELISA
titer, while a positive control (GM-CSF plasmid) increased the titer
(data not shown).
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To determine whether coinjection with a different dose of IL-12
genes influences the survival of animals after lethal challenge with
HSV-2 (186), inbred animals were challenged with 4 x
LD50 5 wk after the initial immunization. As shown in Fig. 9A, naive mice succumbed to
death within 13 days after viral challenge. However, gD DNA vaccine (6
µg) alone increased the survival of mice to 60%. Furthermore,
coinjection with 5 and 40 µg of IL-12 genes increased the survival of
mice to 88 and 80%, respectively. In contrast, coinjection with 20
µg of IL-12 genes resulted in complete protection from lethal
herpetic infection. These data are supportive of dose-response effects
mediated by local cytokine production. Fig. 9B and Table I
also show protective effects of IL-12 genes on HSV-2 challenge in
outbred mice. Mice injected with either pgD backbone plasmids (pAPL-1)
or IL-12 genes (50 µg) displayed 22 and 40% survival of mice,
respectively. However, gD DNA vaccine alone showed 90% survival of
mice 20 days following viral infection, but the survival rate was
decreased to 70% thereafter. Furthermore, complete protection from
HSV-2 challenge was observed by coinjecting IL-12 genes (50 µg).
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It has been reported that Th1-type CD4+ T cells, but
not CD8+ T cells, are responsible for protecting animals
from HSV challenge 17, 26, 27 . In our studies we also investigated
the induction of gD-specific CTL responses by gD DNA vaccination, but
no CTL activity was observed. Furthermore, no CTL data have been
reported after immunization with gD DNA vaccine 19, 28, 29 . Thus, we
next focused on possible roles of CD4+ T cells in inducing
IL-12-enhanced protective immunity against viral infection. As shown in
Fig. 10, all animals immunized with pgD
plus IL-12 survived lethal HSV challenge. However, vaccinated animals
treated with anti-CD4 Abs failed to survive lethal challenge in a
manner similar to control animals. The data support the idea that IL-12
can enhance survival through effects on CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA vaccination has been shown to induce both humoral and cellular immune responses to Ags of interest and then confer protective immunity against certain viral pathogens in animal models 17, 37, 38, 39, 40, 41 . Furthermore, in DNA vaccination studies a variety of cytokine genes have been applied to induce better protective immunity against viral infection. For example, GM-CSF genes when coinjected with DNA vaccines increased survival in both a rabies mouse model as well as an encephalomyocarditis virus mouse model 39, 42 . A number of studies have reported enhancement of cellular or humoral responses after cytokine codelivery 13, 43, 44, 45 . In particular, coinjection of IL-12 cDNA enhances Ag-specific T cell proliferation responses and CTL activities in DNA vaccine studies 13, 14 . However, these collective studies did not demonstrate what effects if any such vaccination would have in any pathogenic challenge. Furthermore, it would not be surprising if different pathogens would behave uniquely due to different immune correlates. In the present study we observed significant enhancement of protective cell-mediated immune responses with regard to the HSV model when combining a DNA vaccine for gD with the IL-12 cDNAs.
Direct i.m. injection of IL-12 genes resulted in increased expression
of the Ly6 molecule on splenocytes. Furthermore, IL-12-mediated effects
on Ly6 expression were observed up to 80 days after DNA injection. This
suggests that IL-12 molecules are continuously produced by i.m.
injection of IL-12 cDNA, resulting in activating resting immune cells
in vivo. However, significant inhibition of gD-specific Ab (systemic
IgG, IgA, IgE, and local IgA) production was observed through vaccine
modulation by codelivery with IL-12 genes. This is compatible with the
previous finding that coinjection with IL-12 molecules showed a
significant inhibition of IgG responses to a viral DNA vaccine 13 . In
contrast, increased Th cell proliferation was achieved by coinjecting
plasmid DNAs encoding IL-12. This is again compatible with the previous
finding that coinjection with IL-12 molecules increases Ag-specific Th
cell proliferation 13 . This pattern is in line with cytokine
production levels showing that coinjection with IL-12 cDNA enhanced
both IL-2 and IFN- secretion but inhibited IL-4 production. This
implies that IL-12 gene delivery was capable of activating T cells that
generate Th1-type cytokines (IL-2 and IFN-) but inactivate T cells
secreting Th2-type cytokines (IL-4). Similarly, IL-12 coinjection
appears to be inhibitory to the production of Th2-type cytokine
(IL-10). Moreover, Th1 cell-mediated ocular inflammatory disease by HSV
infection was suppressed by topical administration of IL-10
protein, resulting from suppressed chemokine production 46 . The
disease (inflammation in the eye) was also ameliorated by injection
with anti-MIP-1, but not anti-MCP-1, indicating that
MIP-1 might be related to induction of Th1 type cell-mediated immune
responses. In our study we also observed that IL-12 coinjection
enhanced the production of RANTES and MIP-1, but not MCP-1, from
splenocytes after in vitro stimulation with gD. This indicates that
RANTES and MIP-1 might be involved in the induction of strong
Th1-type cellular immune responses 47 . In these cases this activity
cannot be ascribed to backbone CpG motifs, as mixing of gD plasmids
with pCDNA3 vector did not demonstrate similar immune modulatory
function or a change in challenge outcome (data not shown). Thus, the
application of IL-12 cDNA in DNA vaccine studies appears to be
warranted for inducing Ag-specific Th1-type cell-mediated immune
responses.
It has been reported that humoral, cellular, or both immune responses could be responsible for protective immunity against HSV infection 31, 48 . During viral infection, neutralizing Abs can inactivate free viral particles, but are not able to inhibit intracellular HSV infection 49 . When immunized passively with anti-HSV serum, an inhibition of local viral replication was observed in T cell-depleted animals 50 . It appears that Ab-dependent, complement-mediated, and Ab-dependent cell-mediated cytotoxicity are involved in the prevention of HSV infection 49, 51, 52, 53 . Furthermore, it has been suggested that HSV-specific cellular-mediated immunity may play an important major effector function to eradicate HSV-infected cells and control recurrent HSV infection 16, 49, 54, 55 .
We observed that IL-12 cDNA injection alone showed a minor beneficial
effect on both mortality and morbidity resulting from HSV-2 infection.
Further coinjection of pgD and IL-12 significantly induced protection
from morbidity in both inbred and outbred mice. Furthermore, less
herpetic lesions and faster recovery from the herpetic lesions were
achieved by coinjecting IL-12 cDNA with gD Ag in both inbred and
outbred mice. In the case of mortality, coinjection with IL-12 genes
(50 µg) significantly enhanced survival rates of mice after HSV
infection in outbred mice compared with the effect of injecion of the
negative plasmid control and the IL-12 gene alone. This is also
compatible with our results of inbred mouse challenge studies showing
that coinjection with different doses of IL-12 cDNA (5, 20, and 40
µg/chain) all enhanced the survival of mice compared with gD DNA
vaccine alone. In particular, coinjection with IL-12 cDNA (20
µg/chain) alone resulted in complete protection from lethal HSV-2
challenge. At the 20-µg dose of IL-12, gD-specific IgG levels were
completely inhibited. This indicates that cell-mediated immune
responses are mainly involved in reducing mortality and morbidity
resulting from HSV infection. This also suggests that the concentration
ratio of DNA vaccines to adjuvants is a factor to be considered for
IL-12 adjuvancy in a herpes mouse model and possibly other viral
infection models. It seems likely that a lower concentration ratio of
DNA vaccine to adjuvant tends to reduce the generation of
vaccine-specific immunity mediated by surplus adjuvants. Taken
together, it seems that there is a positive correlation between
protective immunity and cellular immunity, which is explained by
increased Th cell proliferation responses and cytokine (IL-2 and
IFN-) production levels when coinjected with IL-12 molecules. Our
observation of enhanced cell-mediated immune responses is supported by
previous findings that coinjection with IL-12 cDNA has a potent effect
on the induction of Th cell proliferation and CTL activity in HIV and
influenza DNA vaccine studies 13, 14 . Recently, it has been reported
that Th1-type CD4+ T cells, but not CD8+ T
cells, are responsible for protecting animals from HSV challenge 17, 26, 27 . In our studies, we also investigated the induction of
gD-specific CTL responses by gD DNA vaccination, but no CTL activity
was observed by us here (data not shown), which is consistent with
previous findings 19, 28, 29 . These other studies suggested that gD
might not possess a CTL epitope in this particular haplotype. This
suggests that the protective effect of IL-12 genes is dependent on
CD4+ T cell-mediated immunity. We directly confirmed this
hypothesis through in vivo deletion of the CD4+ T cell
subset using anti-CD4 Ab treatment of mice. In vivo CD4 deletion
suppressed the IL-12-enhanced protective immunity against HSV
challenge. We also observed that coinjection with IL-12 cDNA increased
IFN- production in vitro. IFN- plays a critical role in host
resistance directly or via induction of an anti-HSV state in
lymphocytes and macrophages 56, 57, 58, 59, 60, 61 . Taken together, it seems that
protective effects of IL-12 gene coinjection might be to stimulate a
more Th1-type CD4+ T cell.
In conclusion, the data presented here suggest that IL-12-induced cellular immune responses, in particular Th1-type CD4+ T cell-mediated responses, mediate enhanced protection from mortality and morbidity against lethal HSV-2 challenge in gD DNA vaccine studies. Moreover, better protection could be achieved by injecting both DNA vaccine and IL-12 cDNA at the proper concentration, suggesting that the dosage between vaccine and IL-12 is a factor to be considered in DNA vaccine trials. These studies also demonstrate that molecular adjuvants can increase both the potency and the focus of vaccine preparations, indicating the need for additional study of such multicomponent preparations for both vaccine and immune therapeutic applications.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David B. Weiner, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: HSV, herpes simplex virus; GM-CSF, granulocyte macrophage CSF; HRP, horseradish peroxidase; MCP-1, monocyte chemotactic protein-1; MIP-1, macrophage inflammatory protein-1; LD50, half-maximal lethal dose.
Received for publication May 8, 1998. Accepted for publication December 3, 1998.
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A. Smith, F. Santoro, G. Di Lullo, L. Dagna, A. Verani, and P. Lusso Selective suppression of IL-12 production by human herpesvirus 6 Blood, October 15, 2003; 102(8): 2877 - 2884. [Abstract] [Full Text] [PDF]
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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]
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L. G. Bermudez-Humaran, P. Langella, N. G. Cortes-Perez, A. Gruss, R. S. Tamez-Guerra, S. C. Oliveira, O. S. Cardenas, R. Montes de Oca-Luna, and Y. Le Loir Intranasal Immunization with Recombinant Lactococcus lactis Secreting Murine Interleukin-12 Enhances Antigen-Specific Th1 Cytokine Production Infect. Immun., April 1, 2003; 71(4): 1887 - 1896. [Abstract] [Full Text] [PDF]
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 H.-W. Chen, Y.-P. Lee, Y.-F. Chung, Y.-C. Shih, J.-P. Tsai, M.-H. Tao, and C.-C. Ting Inducing long-term survival with lasting anti-tumor immunity in treating B cell lymphoma by a combined dendritic cell-based and hydrodynamic plasmid-encoding IL-12 gene therapy Int. Immunol., March 1, 2003; 15(3): 427 - 435. [Abstract] [Full Text] [PDF]
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 M. Zhu, X. Xu, H. Liu, X. Liu, S. Wang, F. Dong, B. Yang, and G. Song Enhancement of DNA vaccine potency against herpes simplex virus 1 by co-administration of an interleukin-18 expression plasmid as a genetic adjuvant J. Med. Microbiol., March 1, 2003; 52(3): 223 - 228. [Abstract] [Full Text] [PDF]
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D. M. Koelle and L. Corey Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research Clin. Microbiol. Rev., January 1, 2003; 16(1): 96 - 113. [Abstract] [Full Text] [PDF]
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 T.-Y. Kim, H.-J. Myoung, J.-H. Kim, I.-S. Moon, T.-G. Kim, W.-S. Ahn, and J.-I. Sin Both E7 and CpG-Oligodeoxynucleotide Are Required for Protective Immunity against Challenge with Human Papillomavirus 16 (E6/E7) Immortalized Tumor Cells: Involvement of CD4+ and CD8+ T Cells in Protection Cancer Res., December 15, 2002; 62(24): 7234 - 7240. [Abstract] [Full Text] [PDF]
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 D. Huang, M. Tani, J. Wang, Y. Han, T. T. He, J. Weaver, I. F. Charo, V. K. Tuohy, B. J. Rollins, and R. M. Ransohoff Pertussis Toxin-Induced Reversible Encephalopathy Dependent on Monocyte Chemoattractant Protein-1 Overexpression in Mice J. Neurosci., December 15, 2002; 22(24): 10633 - 10642. [Abstract] [Full Text] [PDF]
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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., October 15, 2002; 76(20): 10540 - 10545. [Abstract] [Full Text] [PDF]
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U. Palendira, A. T. Kamath, C. G. Feng, E. Martin, P. J. Chaplin, J. A. Triccas, and W. J. Britton Coexpression of Interleukin-12 Chains by a Self-Splicing Vector Increases the Protective Cellular Immune Response of DNA and Mycobacterium bovis BCG Vaccines against Mycobacterium tuberculosis Infect. Immun., April 1, 2002; 70(4): 1949 - 1956. [Abstract] [Full Text] [PDF]
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 K. R. B. Bastos, J. M. Alvarez, C. R. F. Marinho, L. V. Rizzo, and M. R. D'Imperio Lima Macrophages from IL-12p40-deficient mice have a bias toward the M2 activation profile J. Leukoc. Biol., February 1, 2002; 71(2): 271 - 278. [Abstract] [Full Text] [PDF]
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 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]
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G. Reiner, E. Melchinger, M. Kramarova, E. Pfaff, M. Buttner, A. Saalmuller, and H. Geldermann Detection of quantitative trait loci for resistance/susceptibility to pseudorabies virus in swine J. Gen. Virol., January 1, 2002; 83(1): 167 - 172. [Abstract] [Full Text] [PDF]
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R. Garcia-Navarro, B. Blanco-Urgoiti, P. Berraondo, R. Sanchez de la Rosa, A. Vales, S. Hervas-Stubbs, J. J. Lasarte, F. Borras, J. Ruiz, and J. Prieto Protection against Woodchuck Hepatitis Virus (WHV) Infection by Gene Gun Coimmunization with WHV Core and Interleukin-12 J. Virol., October 1, 2001; 75(19): 9068 - 9076. [Abstract] [Full Text] [PDF]
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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]
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P. C. Melby, J. Yang, W. Zhao, L. E. Perez, and J. Cheng Leishmania donovani p36(LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis Infect. Immun., August 1, 2001; 69(8): 4719 - 4725. [Abstract] [Full Text] [PDF]
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 D. Stober, R. Schirmbeck, and J. Reimann IL-12/IL-18-Dependent IFN-{{gamma}} Release by Murine Dendritic Cells J. Immunol., July 15, 2001; 167(2): 957 - 965. [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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P. Harle, S. Noisakran, and D. J. J. Carr The Application of a Plasmid DNA Encoding IFN-{{alpha}}1 Postinfection Enhances Cumulative Survival of Herpes Simplex Virus Type 2 Vaginally Infected Mice J. Immunol., February 1, 2001; 166(3): 1803 - 1812. [Abstract] [Full Text] [PDF]
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D. J. Shedlock and D. B. Weiner DNA vaccination: antigen presentation and the induction of immunity J. Leukoc. Biol., December 1, 2000; 68(6): 793 - 806. [Abstract] [Full Text]
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J.-I. Sin, J. J. Kim, C. Pachuk, C. Satishchandran, and D. B. Weiner DNA Vaccines Encoding Interleukin-8 and RANTES Enhance Antigen-Specific Th1-Type CD4+ T-Cell-Mediated Protective Immunity against Herpes Simplex Virus Type 2 In Vivo J. Virol., December 1, 2000; 74(23): 11173 - 11180. [Abstract] [Full Text] [PDF]
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C. M. Leutenegger, F. S. Boretti, C. N. Mislin, J. N. Flynn, M. Schroff, A. Habel, C. Junghans, S. A. Koenig-Merediz, B. Sigrist, A. Aubert, et al. Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif J. Virol., November 15, 2000; 74(22): 10447 - 10457. [Abstract] [Full Text] [PDF]
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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]
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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]
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J.-I. Sin, V. Ayyavoo, J. Boyer, J. Kim, R. B. Ciccarelli, and D. B. Weiner Protective immune correlates can segregate by vaccine type in a murine herpes model system Int. Immunol., November 1, 1999; 11(11): 1763 - 1773. [Abstract] [Full Text] [PDF]
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A. Lobell, R. Weissert, S. Eltayeb, C. Svanholm, T. Olsson, and H. Wigzell Presence of CpG DNA and the Local Cytokine Milieu Determine the Efficacy of Suppressive DNA Vaccination in Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 1999; 163(9): 4754 - 4762. [Abstract] [Full Text] [PDF]
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