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*
Beth Israel Deaconess Medical Center, Boston, MA 02215;
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; and
Merck Research Laboratories, West Point, PA 19486
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid DNA vaccines have been shown to elicit CTL, Th cell, and Ab responses in a variety of animal models (6, 7, 8, 9). The potential utility of plasmid DNA as a candidate HIV-1 vaccine strategy has therefore been an area of active investigation (10, 11, 12, 13, 14, 15, 16, 17, 18). DNA vaccines augmented by IL-2/Ig cytokine fusion constructs as well as DNA/MVA prime-boost regimens have recently been shown to elicit potent virus-specific CTL responses and to provide substantial control of viral replication following a pathogenic SHIV challenge in rhesus monkeys (19, 20). Optimizing the induction of vaccine-elicited HIV-specific CD4+ T lymphocyte responses may further improve candidate HIV-1 vaccine strategies.
Plasmid GM-CSF has been investigated as a potential vaccine adjuvant in a number of murine disease models. Coadministration of plasmid GM-CSF was first shown to augment the Ab response elicited by a rabies-specific DNA vaccine (21). Plasmid GM-CSF has subsequently been shown to increase DNA vaccine-elicited immune responses, including cellular proliferative responses to HIV-1 (22, 23, 24), hepatitis C virus (25, 26), herpes simplex virus type 2 (27), Mycobacterium tuberculosis (28), and Plasmodium yoelii (29). The mechanism for the adjuvant properties of plasmid GM-CSF may involve increased recruitment of macrophages and dendritic cells to the site of injection (30, 31, 32).
In this report we investigate the ability of plasmid GM-CSF to augment the CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. We show that the delivery of a bicistronic plasmid expressing both gp120 and GM-CSF elicits dramatic gp120-specific CD4+ T lymphocyte responses. Precise temporal and spatial codelivery of Ag and GM-CSF therefore appears to be critical for optimal induction of DNA vaccine-elicited immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasmid DNA vaccine pV1J-gp120 expressing HIV-1 IIIB gp120 was used for these experiments (33). Construction of the monocistronic pV1J-GM-CSF and bicistronic pV1J-gp120/GM-CSF plasmids has been described previously (34). In the bicistronic pV1J-gp120/GM-CSF plasmid an internal ribosome entry site from encephalomyocarditis virus was placed between the two genes to obtain efficient internal initiation of translation (35). The sham plasmid was the empty pV1J vector. Plasmids were prepared from large scale bacterial cultures by standard alkaline lysis followed by double CsCl gradient banding (34).
Mice and immunizations
Eight- to 12-wk-old BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). Groups of mice (n = 4/group) were immunized with varying concentrations of plasmid DNA in 50 µl 0.15 M sterile saline. Injections were performed i.m. in the quadriceps muscle. For experiments with recombinant vaccinia virus, 5 x 107 PFU vac-HIV-1 Env BH-10 gp160 (Therion Biologics, Cambridge, MA) was injected i.p. in 200 µl sterile PBS. For both DNA and vaccinia immunizations, the seroconversion rate was 100% at 2 wk. Mice were typically sacrificed for immunological assays at 3 wk.
Antibody ELISA
Serum anti-gp120 Ab titers from immunized mice were measured by a direct ELISA as previously described (34). Ninety-six-well plates coated overnight with 100 µl/well of 1 µg/ml recombinant IIIB gp120 (Intracel, Cambridge, MA) in PBS were blocked for 2 h with PBS containing 2% BSA and 0.05% Tween 20. Sera were then added in serial dilutions and incubated for 1 h. The plates were washed three times with PBS containing 0.05% Tween 20 and incubated for 1 h with a 1/5000 dilution of a peroxidase-conjugated affinity-purified rabbit anti-mouse secondary Ab (Jackson ImmunoResearch Laboratories, Bar Harbor, ME). The plates were then washed three times, developed with tetramethylbenzidine (Kirkegaard & Perry, Gaithersburg, MD), stopped with 1% HCl, and analyzed at 450 nm with a Dynatech MR5000 ELISA plate reader.
Tetramer staining assays
Tetrameric H-2Dd complexes folded around
the HIV-1 IIIB V3 loop optimal P18 epitope peptide (P18-I10 or
RGPGRAFVTI) (36) were prepared and used to stain
P18-specific CD8+ T cells essentially as
previously described (37, 38). Mouse blood was collected
in RPMI 1640 containing 40 U/ml heparin. Following lysis of the RBCs,
0.1 µg PE-labeled Dd/P18 tetramer in
conjunction with APC-labeled anti-mouse CD8
mAb (Ly-2, Caltag,
San Francisco, CA) were used to stain P18-specific
CD8+ T cells. The cells were washed in PBS
containing 2% FBS and fixed in 0.5 ml PBS containing 1.5%
paraformaldehyde. Samples were analyzed by two-color flow cytometry on
a FACSCalibur (BD Biosciences, Mountain View, CA). Gated
CD8+ T lymphocytes were examined for staining
with the Dd/P18 tetramer. All tetramer staining
experiments were confirmed by standard functional chromium release CTL
assays using P18 peptide-stimulated splenocytes as effector cells as
previously described (34, 38).
Proliferation assays
Standard [3H]thymidine incorporation assays were performed to assess CD4+ T cell proliferative responses. Splenocytes from immunized mice were resuspended at 4 x 106 cells/ml in RPMI 1640 containing 5% FBS. One hundred microliters was added to each well in 96-well plates with 1, 0.2, 0.04, or 0 µg/ml recombinant HIV-1 IIIB gp120 (Intracel, Cambridge, MA). After 4 days of culture 1 µCi [3H]thymidine (ICN Biochemicals, Costa Mesa, CA) was added to each well. Following a 16-h incubation, cells were harvested on glass filter paper, and radioactivity was measured in a Wallac 1450 Microbeta liquid scintillation counter (Wallac, Gaithersburg, MD). The stimulation index (SI)3 was calculated as: (cpm with Ag stimulation)/(background cpm without Ag). For experiments involving depletion of CD4+ or CD8+ T cells, splenocytes were incubated with magnetic microbeads coated with mAbs specific for murine CD4 (L3T4) or CD8 (Ly-2; Miltenyi Biotec, Auburn, CA). Separation using MiniMACS separation columns was performed according to the manufacturer’s instructions. Cell depletions were 95–100% efficient.
ELISPOT assays
ELISPOT assays were used to assess IFN-
production by
unfractionated splenocytes or splenocytes depleted of
CD4+ T cells or CD8+ T
cells. IFN- responses were measured using the optimal CTL epitope
peptide P18 (36) or a pool of 47 overlapping 15-mer
peptides derived from HIV-1 IIIB Env gp120 (Centralized Facility for
AIDS Reagents, Potters Bar, U.K.). Ninety-six-well multiscreen plates
(Millipore, Bedford, MA) coated overnight with 100 µl/well of 10
µg/ml rat anti-mouse IFN- (BD PharMingen, San Diego, CA)
in PBS were washed with endotoxin-free Dulbecco’s PBS (Life
Technologies, Gaithersburg, MD) containing 0.25% Tween 20 and blocked
with PBS containing 5% FBS for 2 h at 37°C. The plates were
washed three times with Dulbecco’s PBS containing 0.25% Tween 20,
rinsed with RPMI 1640 containing 10% FBS, and incubated in triplicate
with 5 x 105 splenocytes/well in a 100-µl
reaction volume containing 8 µg/ml peptide. For studies using the Env
peptide pool, each peptide in the pool was present at 8 µg/ml.
Following an 18-h incubation, the plates were washed nine times with
Dulbecco’s PBS containing 0.25% Tween 20 and once with distilled
water. The plates were then incubated for 16 h with 75 µl/well 5
µg/ml biotinylated rat anti-mouse IFN-, washed six times with
Coulter wash (Coulter, Miami, FL), and incubated for 2.5 h with a
1/500 dilution of streptavidin-AP (Southern Biotechnology Associates,
Birmingham, AL). Following five washes with Coulter wash and once with
PBS, the plates were developed with nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate chromogen (Pierce,
Rockford, IL), stopped by washing with tap water, air-dried, and read
using an ELISPOT reader (Hitech Instruments, Edgement, PA).
Cytokine secretion assays
Splenocytes (4 x 106) in 1 ml RPMI 1640 containing 5% FBS were cultured with 1 µg/ml recombinant HIV-1 IIIB gp120 (Intracel, Cambridge, MA). After 72 h supernatants were harvested and analyzed for the presence of cytokines using commercial ELISA kits according to the manufacturer’s protocols (Endogen, Cambridge, MA).
Pathological analysis
The quadriceps muscles from mice were excised 7 days after plasmid DNA immunizations and frozen in Tissue Freezing Medium (Jung, Nussloch, Germany) in a dry ice/methanol bath. Five-micrometer-thick sections were cut with a cryostat, air-dried, and stained with H&E.
In vivo protein expression assays
To quantitate GM-CSF expressed in vivo, quadriceps muscles from vaccinated mice were excised 48 h after plasmid DNA immunizations. Muscles were then homogenized with No. 10 Medicon homogenizers (BD Biosciences) in 1 ml PBS containing 0.05% Tween 20. Muscle homogenates were incubated on ice for 30 min, cell debris was removed by centrifugation, and supernatants were analyzed for GM-CSF by ELISA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initiated studies by coimmunizing mice with the DNA vaccine pV1J-gp120 expressing HIV-1 IIIB gp120 plus the monocistronic pV1J-GM-CSF plasmid (34). Groups of mice were immunized i.m. with 50 µg sham plasmid, 50 µg pV1J-GM-CSF alone, 50 µg pV1J-gp120 alone, or 50 µg pV1J-gp120 plus 50 µg pV1J-GM-CSF on day -2, 0, or +2 relative to the pV1J-gp120 immunization. Ab, tetramer staining, cytotoxicity, and proliferative responses were measured 3 wk following initial immunization.
As shown in Fig. 1
, A and
B, comparable anti-gp120 Ab responses and P18-specific
CD8+ T lymphocyte tetramer responses were
measured in all the gp120-vaccinated groups. P18-specific
chromium-release CTL cytotoxicity assays using P18-stimulated
splenocytes as effector cells mirrored the results of the tetramer
staining studies (data not shown). As shown in Fig. 1C,
gp120-specific splenocyte proliferative responses were augmented
approximately 2-fold in the animals that were inoculated concurrently
with pV1J-gp120 and pV1J-GM-CSF. The mean SI of splenocytes using 0.2
µg/ml gp120 stimulation was 6 in mice immunized with pV1J-gp120
alone, but was 14 in mice that received pV1J-GM-CSF on day 0 concurrent
with pV1J-gp120 vaccination. Interestingly, no augmentation of this
response was observed when pV1J-GM-CSF was injected on day -2 or day
2. In addition, no adjuvant effect was detected when 50 µg pV1J-gp120
was delivered with 50 µg pV1J sham plasmid control, and no adjuvant
effect was observed when 50 µg pV1J-gp120 and 50 µg pV1J-GM-CSF
were inoculated at different muscle sites (data not shown). These
experiments suggested that augmentation of vaccine-elicited
proliferative responses by plasmid GM-CSF required the precise temporal
and spatial codelivery of GM-CSF with Ag.
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We reasoned that the most precise codelivery of GM-CSF with Ag would be achieved with a bicistronic pV1J-gp120/GM-CSF plasmid that coexpressed gp120 and GM-CSF under control of a single promoter. The gp120/GM-CSF bicistronic plasmid (34) included a single promoter with the two genes separated by the internal ribosome entry site from encephalomyocarditis virus to obtain efficient internal initiation of translation (35).
The immune responses elicited by pV1J-gp120/GM-CSF were compared with
those elicited by the combination of the two monocistronic plasmids and
with those elicited by recombinant vaccinia-env. Groups of
mice were immunized with 50 µg pV1J sham plasmid, 50 µg pV1J-gp120,
50 µg pV1J-gp120 plus 50 µg pV1J-GM-CSF, 50 µg pV1J-gp120/GM-CSF,
or 5 x 107 PFU vac-env. As shown
in Fig. 2, A and B,
comparable anti-gp120 Ab responses and P18-specific
CD8+ T cell tetramer responses were elicited in
the three groups of DNA-vaccinated mice, although significantly higher
responses were observed in the mice immunized with vac-env.
Functional CTL assays performed concurrently corroborated these
tetramer staining results (data not shown).
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C shows the CD4+ T lymphocyte
proliferative responses of splenocytes from these animals. No
gp120-specific proliferative responses were detectable in the mice that
received the pV1J sham vaccine. As before, proliferative responses were
readily measured in the mice that received pV1J-gp120, and 2-fold
higher proliferative responses were observed in the animals that were
injected concurrently with pV1J-gp120 plus pV1J-GM-CSF. Strikingly, the
proliferative responses from mice immunized with the bicistronic
pV1J-gp120/GM-CSF vaccine were approximately 10-fold higher than the
proliferative responses from mice immunized with the monocistronic
pV1J-gp120 (mean SI of 118 compared with 9 using 0.2 µg/ml gp120
stimulation). The proliferative responses primed by pV1J-gp120/GM-CSF
were also markedly higher than those primed by high dose
vac-env immunization. Moreover, the increased proliferative
responses elicited by pV1J-gp120/GM-CSF persisted for >3 mo (data not
shown). As shown in Fig. 2D, depletion of
CD4+ T cells, but not CD8+
T cells, before performing the proliferation assays abrogated these
responses, confirming that the proliferative responses primed by
pV1J-gp120 and pV1J-gp120/GM-CSF were CD4+ T cell
responses.
As shown in Table I, splenocytes from
these animals were then examined for their ability to secrete cytokines
following stimulation with recombinant gp120. Cultured splenocytes from
all the plasmid DNA- and recombinant vaccinia-immunized mice had high
levels of IL-2 and IFN- production and low levels of IL-4 and IL-10
production, consistent with Th1-type immune responses. Compared with
mice immunized with only pV1J-gp120, the addition of the monocistronic
GM-CSF plasmid resulted in marginally higher IL-2 production. In
contrast, splenocytes from mice immunized with the bicistronic
gp120/GM-CSF plasmid demonstrated 3-fold increases in IFN-, IL-2,
IL-4, IL-10, and GM-CSF production, consistent with the increased
proliferative activity observed in the splenocytes of these
animals.
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the monocistronic GM-CSF plasmid had lower GM-CSF expression in vivo in
mouse muscle compared with the bicistronic gp120/GM-CSF plasmid.
Increasing the dose of the monocistronic GM-CSF plasmid, however,
led to increased cytokine expression and increased proliferative
responses. A high dose (500 µg) of the monocistronic GM-CSF plasmid
was required to elicit proliferative responses comparable with a
standard dose (50 µg) of the bicistronic gp/120 GM-CSF plasmid.
Moreover, comparable levels of gp120 were detected from COS cells
transiently transfected with pV1J-gp120 or pV1J-gp120/GM-CSF (data not
shown). These data suggest that the immunogenicity of the bicistronic
gp120/GM-CSF vaccine reflected both efficient cytokine expression as
well as coordinate expression of GM-CSF with gp120.
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, the dose of plasmid required to
elicit detectable proliferative responses (defined as SI >3) was 5
µg for pV1J-gp120, but was only 0.05 µg for pV1J-gp120/GM-CSF.
Thus, the bicistronic gp120/GMCSF vaccine was approximately 100-fold
more potent than the monocistronic gp120 DNA vaccine in eliciting
detectable proliferative responses.
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, only
pV1J-gp120/GM-CSF primed for markedly augmented gp120-specific
splenocyte proliferative responses. Thus, this adjuvant effect was
specific for GM-CSF.
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responses
To confirm the results obtained with the proliferation assays, we
used IFN- ELISPOT assays to measure gp120-specific T cell responses
of splenocytes from vaccinated mice. We measured
CD4+ T cell and CD8+ T cell
IFN- ELISPOT responses by depleting splenocytes of lymphocyte
subsets before peptide stimulation. Mice were immunized with 50 µg
pV1J sham plasmid, 50 µg pV1J-gp120, or 50 µg pV1J-gp120/GM-CSF.
Fig. 5A shows the results of
ELISPOT assays in which splenocytes were stimulated with a pool of 47
overlapping 15-mer peptides spanning the entire gp120 IIIB protein.
Splenocytes from sham-vaccinated mice had no detectable IFN-
responses (zero to five spots per 106 cells).
Potent IFN- responses were detected in splenocytes from mice
immunized with pV1J-gp120, and higher total IFN- responses were
observed in mice immunized with pV1J-gp120/GM-CSF. Following depletion
of CD4+ T cells, comparable IFN- responses
were observed in both groups of mice, suggesting that both vaccines
elicited comparable CD8+ T cell responses.
However, following depletion of CD8+ T cells,
IFN- responses from mice immunized with pV1J-gp120/GM-CSF were
>7-fold higher than from mice immunized with pV1J-gp120, confirming
that the gp120/GM-CSF bicistronic vaccine elicited markedly augmented
CD4+ T cell responses. Fig. 5B shows
the results of ELISPOT assays in which splenocytes were stimulated with
the single optimal H-2Dd-restricted CTL epitope
peptide P18 (RGPGRAFVTI) (36). Similar P18-specific
IFN- responses were detected in splenocytes from mice immunized with
pV1J-gp120 and pV1J-gp120/GM-CSF. Depletion of
CD8+ T cells abrogated these responses,
confirming that both vaccines elicited comparable P18-specific
CD8+ T cell responses.
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We next examined the cellular infiltrates at the site of DNA
inoculation. Mice were injected i.m. with 50 µg pV1J sham plasmid, 50
µg pV1J-gp120, or 50 µg pV1J-gp120/GM-CSF and were sacrificed after
7 days for histologic analysis of the injected quadriceps muscles.
Numerous sequential sections were analyzed. Fig. 6 shows representative H&E-stained
sections. Whereas the pV1J sham plasmid evoked little or no
inflammatory response, inoculation with pV1J-gp120 evoked moderate
cellular infiltrates consisting of 40–100
cells/µm2. In contrast, injection with
pV1J-gp120/GM-CSF evoked dense inflammatory responses consisting of
300–400 cells/µm2, with large clusters of
inflammatory cells as well as scattered cells throughout the
interstitium. These cellular infiltrates were comprised predominantly
of macrophages, CD4+ T cells, and neutrophils, as
determined by immunohistochemistry (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A modest augmentation of CD4+ T lymphocyte responses was observed by coinjecting the monocistronic pV1J-GM-CSF plasmid together with the pV1J-gp120 DNA vaccine. This effect could be amplified by increasing the dose of pV1J-GM-CSF, but it required simultaneous temporal and spatial delivery of both plasmids. A previous study has similarly shown that separating the delivery of plasmid GM-CSF from the plasmid Ag abrogated the adjuvanticity of GM-CSF (32). These observations, together with the dramatic augmentation of CD4+ T cell responses elicited by the bicistronic pV1J-gp120/GM-CSF vaccine, suggest that precise temporal and spatial codelivery of Ag and GM-CSF is required for optimally harnessing the adjuvant properties of GM-CSF.
Histologic analysis revealed strikingly dense cellular infiltrates in muscles injected with pV1J-gp120/GM-CSF. Smaller infiltrates were detected in muscles inoculated with pV1J-gp120. These infiltrates consisted predominantly of macrophages, CD4+ T cells, and neutrophils (data not shown). Thus, GM-CSF may act by recruiting inflammatory cells to the site of inoculation. These results extend prior studies that have described cellular infiltrates in muscles injected with monocistronic plasmids expressing GM-CSF (30, 31, 32). It is likely that the coordinate expression of Ag and GM-CSF achieved using the bicistronic plasmid approach optimally harnesses these recruited inflammatory cells for maximizing Ag presentation and triggering of CD4+ T cells.
A number of laboratories have reported the ability of plasmid GM-CSF to augment the immunogenicity of DNA vaccines in a variety of experimental models. These reports include increased Ab responses (21, 23, 25, 26, 27, 29), CTL responses (22, 39), and T cell proliferative responses (23, 25, 26, 27, 28, 29). A recent study also showed that the timing of plasmid GM-CSF administration determined the type of immune responses that were augmented (31). The differences in the findings among these studies are significant and may reflect the differences in Ags, expression vectors, and specific assays used. In the system used in the present study, plasmid GM-CSF had a largely selective effect on augmenting CD4+ T cell responses, with little effect on Ab and CTL responses. It is possible that the Ab and CTL responses were not limited by T cell help in our system. A threshold level of T cell help may be required for maximizing Ab and CTL responses, above which little benefit is gained.
The ability of GM-CSF bicistronic DNA vaccines to elicit remarkably potent HIV-specific CD4+ T cell responses in addition to CD8+ T cell responses may be particularly useful in improving the efficacy of prophylactic and therapeutic HIV-1 DNA vaccines. Moreover, Ag/GM-CSF bicistronic constructs could be readily incorporated into recombinant live vector vaccines to augment their immunogenicity as well. During primary HIV-1 infection, the early loss of HIV-specific CD4+ T cells is probably due to their particular susceptibility to infection, and the subsequent deficiency of HIV-specific T cell help may be central to the ultimate failure of the immune system to control viremia (3, 4, 40). Augmented vaccine-elicited CD4+ T cell responses may enhance the control of viremia following HIV-1 infection through a number of immunologic mechanisms, including increasing the proliferation, maturation, and functional activity of CD8+ CTL, providing increased help for B cells, and directly producing antiviral cytokines (40). However, it is theoretically possible that increased numbers of vaccine-elicited CD4+ T cells may instead merely provide a larger number of cellular targets for the virus and accordingly worsen the clinical outcome of HIV-1 infection. Vaccine studies in nonhuman primates will therefore be required to evaluate the efficacy of this GM-CSF bicistron strategy in controlling a pathogenic viral challenge.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dan H. Barouch, Beth Israel Deaconess Medical Center, Research East 113, 330 Brookline Avenue, Boston, MA 02215. E-mail address: dan_barouch{at}hotmail.com
3 Abbreviation used in this paper: SI, stimulation index.
Received for publication May 30, 2001. Accepted for publication November 13, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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