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Journal of Virology, March 1999, p. 2280-2287, Vol. 73, No. 3
Immunology Section, Department of Oncology
and Surgical Sciences, University of Padova, 35128 Padua, Italy
Received 10 August 1998/Accepted 16 November 1998
The intramuscular inoculation of Moloney murine sarcoma/leukemia
(M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo
spontaneous regression due to the induction of a strong immune reaction
mediated primarily by cytotoxic T lymphocytes (CTL). We used a
DNA-based vaccination approach to dissect the CTL response against the
Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to
evaluate whether plasmid DNA-immunized mice would be protected against
a subsequent challenge with syngeneic tumor cells expressing the viral
antigens. Intramuscular DNA vaccination induced CTL against both Gag
and Env proteins. A detailed analysis of epitopes recognized by CTL
generated in mice inoculated with the whole virus and with the
Gag-expressing plasmid confirmed the presence of an immunodominant
peptide in the leader sequence of Gag protein (Gag85-93,
CCLCLTVFL) that is identical to that described in B6 mice immunized
with Friend MuLV-induced leukemia cells. Moreover, CTL generated by
immunization with the Env-encoding plasmid recognized a subdominant Env
peptide (Env189-196, SSWDFITV), originally described in
the B6.CH-2bm13 mutant strain. B6 mice immunized with the
Gag-expressing plasmid were fully protected against a lethal tumor
challenge with M-MuLV-transformed MBL-2 leukemia cells, while
vaccination with the Env-expressing plasmid resulted in rejection of
the tumor in 44% of the mice and in increased survival of an
additional 17% of the animals. Taken together, these results indicate
the existence of a hierarchy in the capacity of different structural
viral proteins to induce a protective immune response against
retrovirus-induced tumors.
Lymphoma and leukemia cell lines
originally induced by the antigenically related Friend, Moloney, and
Rauscher (FMR) murine leukemia viruses (MuLV) have been widely used to
study the role of the T-cell-mediated immune response in the
eradication of advanced disseminated malignancies (9, 20,
28). The importance of cellular immunity in controlling tumor
growth has also been studied in physiologic models represented by the
natural oncogenic process that follows FMR virus infection (11,
12, 21).
In particular, Moloney murine sarcoma virus (M-MSV) is a
replication-defective, acutely transforming retrovirus whose
replication defect can be overcome through the helper activity of
chronic transforming M-MuLV, which encodes the viral envelope
components that are necessary for cell infection. Intramuscular
injection of the M-MSV/M-MuLV complex gives rise to sarcomas that
develop at the inoculation site after a short latency period and
regress spontaneously following the induction of a strong immune
reaction, which is mediated primarily by cytotoxic T lymphocytes (CTL)
(13-15).
As recently reviewed by Hasenkrug and Chesebro (21), studies
aimed at identifying antigenic epitopes recognized by virus-specific effector cells have focused mainly on products of the gag
and env structural genes (16, 22, 32).
In analyses of mice vaccinated with recombinant vaccinia virus
expressing F-MuLV env and gag genes,
CD4+ cells were shown to be activated by immunization with
an Env-expressing construct while CTL were activated by immunization
with a Gag-expressing construct (20, 23). Their recognition
by different T-cell subsets suggested that the gag and
env gene products might undergo different antigen processing
and presentation pathways (20). However, recent reports
demonstrated that both env- and gag-encoded proteins of FMR-MuLV contained immunogenic peptides that could associate with both class I and class II major histocompatibility complex (MHC) molecules and thus were able to activate both CTL and
CD4+ cells (21).
To investigate the capacity of different M-MSV/M-MuLV proteins to
induce CTL generation and confer protection against challenge with
leukemia cells bearing the relevant antigens, we took advantage of
DNA-based immunization, a recently developed procedure based on the
intramuscular or intradermal transfer of a plasmid DNA expression
vector coding for a specific antigen (8, 34). This
vaccination approach affords long-lasting induction of a cellular as
well as a humoral immune response against both infectious agents and
tumor-associated antigens (18). We recently demonstrated that mice immunized with a plasmid expressing the tumor-specific antigen P1A generated a strong CTL response and were protected against
a subsequent challenge with tumor cells expressing the relevant antigen
(30).
In the present study, we evaluated the CTL response induced in B6 mice
following DNA vaccination with plasmids encoding the M-MuLV
gag and env genes in comparison with that induced
following injection of the M-MSV/M-MuLV retroviral complex and carried
out a detailed analysis of epitopes recognized by virus-specific CTL. We also tested whether the immunization achieved was capable of protecting against challenge with a tumor cell line expressing the
viral antigens as tumor-associated antigens. The results showed that
immunization with Gag-expressing plasmid mimicked the immune response
to virus infection, in terms of both CTL generation and protection
against tumor growth; although DNA vaccination with the Env-expressing
vector was effective in generating CTL, it did not confer complete
protection against tumor challenge.
Mice.
Female C57BL/6 (B6) mice, 5 to 6 weeks old, were
purchased from Charles River Laboratories (Calco, Como, Italy).
Procedures involving animals and their care conformed with
institutional guidelines that comply with national and international
laws and policies (EEC Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals,
NIH Publication 85-23, 1985; UKCCR Guidelines for the Welfare of
Animals in Experimental Neoplasia [35]). Mice
used for the in vivo tumor growth experiments were examined daily, and
in compliance with our normal practice, premoribund animals were
sacrificed by being given an ethyl ether overdose.
Tumor cell lines.
MBL-2 is a leukemia cell line
(H-2b) derived from an M-MuLV-infected B6 mouse;
EL-4 Plasmids.
The gag gene of M-MuLV was derived from
pMov-9, a biologically active molecular clone of M-MuLV in pBR322
(19), as an EagI-Asp718 I^
restriction fragment and cloned into intermediate plasmid vectors. The
insert was then transferred to the eukaryotic expression vector pcDNA3
(Invitrogen BV, Leek, The Netherlands) as an
EcoRI-XbaI fragment, resulting in
pcDNA3-gag, in which the gag coding sequence spans nucleotides 346 to 2554 of the viral genome. Plasmid
pCMV-ecoenv-bpA (a gift of N. Somia, The Salk Institute for
Biological Studies, La Jolla, Calif.) is based on Bluescript KS+
(Stratagene, La Jolla, Calif.) and contains the human cytomegalovirus
early promoter, the ecotropic env gene of M-MuLV, and the
polyadenylation signal/site of the bovine growth hormone gene. Mock
plasmid pCMV-eco DNA immunization and virus inoculation protocols.
Mice were
anesthesized by ethyl ether inhalation and injected intramuscularly
(i.m.) three times at 20-day intervals with 100 µg of plasmid in 100 µl of saline solution (50 µl was injected into each tibialis
anterior muscle). Mice that underwent complete tumor regression
following i.m. injection of 100 µl of cell extract containing
defective M-MSV copelleted with its natural helper M-MuLV served as
positive controls for CTL production. The M-MSV/M-MuLV cell extract was
prepared from primary sarcomas induced by serial passages in 1-week-old
BALB/c mice, which had an in vitro M-MSV titer of 3 × 105 PFU/ml on 3T3/FL cells.
MLTC and MLPC.
At 20 days after the last inoculation with
plasmid DNA, spleens were removed and 2.5 × 107
splenocytes were restimulated in vitro in a mixed leukocyte tumor culture (MLTC) with 106 syngeneic irradiated (60 Gy) MBL-2
cells or in a mixed leukocyte peptide culture (MLPC) with peptides
corresponding to amino acids 85 to 93 of
gPr80gag (CCLCLTVFL [10]) or
amino acids 189 to 196 of gp70env (SSWDFITV
[32]). Peptides were synthesized and purified by Tecnogen (Piana di Monte Verna, Caserta, Italy), dissolved to 1 mM in
dimethyl sulfoxide (stock solution), and then diluted in tissue culture
medium to a final concentration of 1 µM. The cultures were set up in
15 ml of DMEM-10% FBS, maintained in 25-cm2 tissue
culture flasks (Falcon, Becton Dickinson, Lincoln Park, N.J.) for 5 days at 37°C under 5% CO2, and then tested for their lytic activity in a 51Cr release assay.
Analysis of CTLp frequency in limiting-dilution assays and
generation of specific CTL clones.
Thirty replicate microcultures
containing various numbers of responder spleen cells were stimulated
with 3 × 104 irradiated (60 Gy) MBL-2 cells and
3 × 105 irradiated (30 Gy) syngeneic spleen cells in
200 µl of DMEM-10% FBS supplemented with 40 U of recombinant
interleukin-2 (Boehringer) per ml. On day 7, the cultures were divided
into three aliquots and tested for cytotoxic activity in
51Cr release assays (see below) with MBL-2,
EL-4 51Cr release assay.
Cytolytic activity was
tested by using a short-term incubation assay. Briefly, 2 × 103 51Cr-labeled target cells were incubated
with effector cells at various effector-to-target-cell ratios in
96-well microplates. After 4 or 6 h of incubation at 37°C,
supernatants were harvested and radioactivity was counted in a
microplate scintillation counter (Top-Count; Packard Instrument Co.,
Meriden, Conn.). For peptide pulsing, 106
51Cr-labeled 293Db, 293Kb or
EL-4 Tumor protection assay.
At 5 weeks after the last DNA
immunization, B6 mice were challenged subcutaneously (s.c.) with 2 × 105 MBL-2 cells and then monitored for a total of 120 days after tumor inoculation. Mice injected with the mock plasmid and
nonimmunized animals served as negative controls. Statistical analysis
of differences among experimental groups was carried out by the
Mantel-Haenszel test.
The Gag85-93 peptide is the immunodominant epitope
recognized by CTL from M-MSV/M-MuLV-induced tumor regressor mice.
It was recently reported that most CTL from B6 mice immunized with
FBL-3 tumor cells, a murine leukemia cell line originally induced by
F-MuLV, recognize a single antigenic peptide (CCLCLTVFL), which is
localized within the leader sequence and has been mapped to amino acids
85 to 93 of gPr80gag, in the transmembrane
domain of the protein (10). This antigenic epitope is
restricted by the H-2Db molecule and is shared
by FMR-induced leukemias (5, 10). To evaluate whether this
peptide represents the major epitope recognized by CTL of B6 mice
inoculated with M-MSV/M-MuLV, we obtained splenocytes from mice in
which an M-MSV/M-MuLV-induced sarcoma had regressed after retroviral
complex inoculation (referred to hereafter as tumor regressor mice) and
performed limiting-dilution analysis to compare the frequency of CTLp
specific for M-MuLV antigen(s) and for the relevant nonapeptide. As
shown in a representative experiment (Fig.
1), we detected a high frequency of CTLp
against FMR-MuLV-negative EL-4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Dissecting the Immune Response to Moloney Murine
Sarcoma/Leukemia Virus-Induced Tumors by Means of a DNA
Vaccination Approach
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
(H-2b) is a chemically
induced thymoma, and EL4+ is a variant infected by and
expressing antigens of FMR-type viruses. 293Db and
293Kb are derivatives of the transformed human embryonic
kidney cell line 293, stably transfected with the murine MHC class I
restriction elements H-2Db and
H-2Kb, respectively (kindly provided by D. Perry-Lalley, Surgery Branch, National Cancer Institute, Bethesda, Md.)
(3). All tumor cell lines were cultured in Dulbecco's
modified Eagle's medium (DMEM) (GIBCO BRL, Paisley, United Kingdom)
supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 µM
2-mercaptoethanol, 150 U of streptomycin per ml, 200 U of penicillin
per ml, and 5 or 10% heat-inactivated fetal bovine serum (FBS) (GIBCO
BRL). 293Db and 293Kb cells were cultured in
the presence of 0.4 mg of G418 (GIBCO) per ml.
was derived from pCMV-ecoenv-bpA by
digestion with XbaI to remove the env gene.
Restriction enzymes were purchased from New England Biolabs (Hitchin,
United Kingdom) and Boehringer (Mannheim, Germany). Plasmid DNA was
purified with Qiagen columns (Qiagen GmbH, Hilden, Germany).
, and Gag85-93-pulsed EL-4
cells as targets. Positive cultures were defined as those in which the
experimental release values exceeded the spontaneous release values by
3 standard deviations (SD). Minimal estimates of CTL precursor (CTLp)
frequencies were calculated from the zero-order term of the Poisson
distribution with linear regression analysis by the least-squares
method (15). Microcultures of spleen cells from mice in
which an M-MSV/M-MuLV-induced sarcoma regressed, as well as from
pcDNA3-gag-injected animals and
pCMV-ecoenv-bpA-injected animals, were also subjected to
limiting-dilution cloning for the generation of specific
CD8+ CTL clones. These clones were restimulated once a week
with irradiated MBL-2 cells and syngeneic spleen cells under the
conditions indicated above and assayed for peptide specificity in
51Cr release assays.
cells per ml were incubated for 30 min at 37°C
with Gag85-93 or Env189-196 peptide at a
final concentration of 0.5 and 5 µM, respectively, and then washed
three times before use.
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
cells pulsed with
Gag85-93 peptide (1 in 2,100), which was comparable to the
frequency of CTLp (1 in 1,700) against MBL-2 tumor cells, a leukemia
cell line derived from an M-MuLV-infected mouse. Moreover, the results
of cold-target competition assays carried out on MLTC bulk cultures
from tumor regressor mice showed that the addition of increasing
numbers of unlabeled MBL-2 or EL-4 cells pulsed with
Gag85-93 peptide reduced the lysis of 51Cr-labeled MBL-2 target cells by a similar extent (data
not shown). Taken together, these data strongly suggest that the
Gag85-93 peptide represented the immunodominant epitope
for CTL generated during the course of the immune response to
M-MSV/M-MuLV virus infection, although we cannot rule out the
possibility that restimulation with MBL-2 leukemia cells does not
reveal some epitopes unique to the defective MSV component.
View larger version (15K):
[in a new window]
FIG. 1.
Frequency of CTLp against the Gag85-93
CCLCLTVFL peptide in tumor regressor B6 mice. Minimal frequencies of
CTLp specific for MBL-2 cells (), Gag85-93
peptide-pulsed EL-4 cells (
), and FMR-MuLV-negative
EL-4 cells (
) were estimated by linear-regression
analysis. Limiting numbers of responder spleen cells from virus-induced
tumor regressor B6 mice were cultivated as reported in Materials and
Methods and divided into three portions for testing in 51Cr
release assays.
CTL generation in B6 mice immunized with plasmid DNA expressing
M-MuLV Gag.
The results described above prompted us to test
whether the injection of plasmid DNA coding for M-MuLV Gag protein
would generate a strong CTL response against the immunodominant
gag-encoded antigen. To this end, mice were inoculated i.m.
three times with 100 µg of pcDNA3-gag plasmid at 20-day
intervals; animals injected with the same vector lacking the insert
(pcDNA3) served as negative controls, while tumor regressor mice were
used as positive controls. At 20 days after the last plasmid DNA
inoculation, splenocytes were stimulated in MLTC with irradiated
syngeneic MBL-2 leukemia cells, and lytic activity was evaluated 5 days
later in a short-term 51Cr release assay. As shown in Fig.
2, all pcDNA3-gag-injected mice generated CTL that efficiently lysed MBL-2 target cells, as well
as EL-4+ cells, a syngeneic leukemia subline expressing
FMR-MuLV-induced antigens. The observation that the parental cell line
EL-4, which lacks the relevant viral antigens, was
resistant to lysis indicated that the cytotoxic activity was virus
specific. As expected, this cytolysis was MHC restricted, since
allogeneic (H-2d) LSTRA target cells expressing
M-MuLV antigens were not killed (data not shown).
|
), and FMR-MuLV-negative
EL-4 cells pulsed with
Gag85-93 but also efficiently lysed EL-4+
target cells expressing the FMR-MuLV antigens. This result suggests that DNA immunization might mimic the natural immune response to viral
infection and hence could provide a useful procedure to define the role
of the different epitope(s) within the M-MSV/M-MuLV sequence that
elicits an efficient CTL response to the virus.
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CTL generation in B6 mice immunized with plasmid DNA expressing M-MuLV Env. It was recently reported that M-MSV/M-MuLV infection in the B6-derived mutant B6.CH-2bm13 strain of mice, whose class I Db antigen-presenting groove is partly shaped by a class I Kb-encoded sequence, induces a strong CTL response against an immunodominant Kb-restricted peptide that spans amino acids 189 to 196 of the M-MuLV Env protein (i.e., SSWDFITV). However, although Env189-196-specific CTL could be induced in normal B6 mice by peptide immunization, the frequency of CTLp recognizing the Kb-restricted SSWDFITV peptide in B6 tumor regressor mice is very low (32).
To assess whether immunization with plasmid DNA expressing Env would also result in the induction of a strong CTL response, B6 mice were injected three times with pCMV-ecoenv-bpA as reported above and sacrificed 20 days after the last plasmid DNA injection, and spleen cells were stimulated in MLTC with MBL-2 cells. Mice receiving repeated injections with the pCMV-eco mock plasmid were used as negative
controls, while tumor regressor mice served as positive controls. As
shown in a representative experiment (Fig.
4), spleen cells from five of seven
pCMV-ecoenv-bpA-injected mice showed high lytic activity
against EL-4+ and MBL-2 target cells, while in two mice the
cytotoxicity was less than 3 SD above the mean value obtained in MLTC
of mock-treated control mice. These results indicate that a DNA-based
immunization approach is capable of expanding a CTL population specific
for an M-MSV/M-MuLV epitope that is poorly recognized in the course of
the virus infection.
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Analysis of MHC restriction of Gag- and Env-specific CTL. To finely dissect the CTL response in tumor regressor and plasmid-inoculated mice and to assess the restriction specificity of the antigenic epitopes recognized by Gag- and Env-specific CTL, the human embryonic cell lines 293Db and 293Kb were pulsed with Gag85-93 and Env189-196 peptides, respectively, and used as targets of CTL generated from spleens of tumor regressor mice or mice injected with the Gag- or Env-expressing plasmids. CTL were generated in MLTC with MBL-2 cells or in MLPC following addition of Gag85-93 (CCLCLTVFL) or Env189-196 (SSWDFITV) antigenic peptides. As reported in Fig. 5, MLTC from tumor regressor mice efficiently lysed both MBL-2 and Gag85-93-pulsed 293Db cells and displayed weak cytotoxic activity against 293Kb cells loaded with Env189-196. However, Env189-196-loaded 293Kb target cells were killed to a much greater extent by MLTC from pCMV-ecoenv-bpA-injected mice; this cytotoxic activity was highly specific, since lysis of MBL-2 cells but not against 293Db cells pulsed with Gag85-93 peptide was also detected. As expected, the cytotoxic activity of MLTC from pcDNA3-gag-injected mice was directed only against the Gag85-93 peptide and was Db restricted. MLTC from animals injected with either of the two mock plasmids did not show lytic activity against any target (data not shown).
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Analysis of CTL clones derived from tumor regressor and
plasmid-injected mice.
We next derived a large panel of CTL clones
from splenocytes of tumor regressor B6 mice by limiting-dilution
cloning with MBL-2 tumor cells as stimulators and tested them for their
specificity against syngeneic virus-transformed cells and
FMR-MuLV-negative Gag85-93 peptide-pulsed tumor cells. Of
97 CTL clones analyzed, 89 (92%) were indeed specific for the
Gag-derived epitope associated with
Db MHC class I, as illustrated by
the results for the representative clone 76GM shown in Fig.
6, while 8 clones killed MBL-2 cells but
not Gag85-93 peptide-pulsed cells. These eight clones were
found to recognize the Env189-196 peptide in the
Kb context (e.g., clone 13/1 [Fig. 6]). These
observations indicate that the few CTL emerging from tumor regressor
mice that were not specific for the immunodominant Gag epitope were
directed against this subdominant Env peptide. Moreover, a set of CTL
clones obtained from mice immunized with pcDNA3-gag
exhibited the same specificity as the majority of CTL clones from tumor
regressor mice and killed MBL-2 cells, EL-4+ cells, and
EL-4 cells pulsed with Gag85-93 peptide but
did not kill EL-4 cells (data not shown).
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Protection against challenge with an M-MuLV-induced MBL-2 leukemia cell line in mice immunized with pcDNA3-gag or pCMV-ecoenv-bpA. Our previous finding that CTL play an essential role in the immune response against M-MSV/M-MuLV-induced tumors led us to evaluate whether CTL generation in mice immunized with plasmid DNA coding for individual structural viral proteins would confer resistance to a subsequent challenge with a lethal dose of tumor cells expressing virus antigens. Therefore, B6 mice vaccinated with pcDNA3-gag were challenged with 2 × 105 MBL-2 leukemia cells 5 weeks after the last immunization; nonimmunized mice and mice injected three times with pcDNA3 vector were used as negative controls. As shown in a representative experiment reported in Fig. 7, all the mice vaccinated with pcDNA3-gag were protected (P < 0.001) while all the nonimmunized and mock-inoculated mice rapidly developed tumors and had to be sacrificed, with mock-injected mice showing a slightly increased survival time compared to nonimmunized mice (P = 0.02). Interestingly, in one experiment, two pcDNA3-gag-immunized mice that had rejected the primary tumor graft developed a secondary tumor that appeared 6 weeks after tumor regression at the site of the original inoculum. However, these tumor cells were not lysed by Gag-specific CTL obtained in MLTC or by CTL clones that specifically recognized the Gag peptide; moreover, cytofluorimetric analysis revealed that these tumor cells were not stained by an anti-gp70 monoclonal antibody (MAb) but expressed Kb and Db molecules at levels comparable to those of the parental MBL-2 cell line (data not shown).
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-injected mice. The
pCMV-ecoenv-bpA-immunized mice showed increased survival compared to the nonimmunized and mock-injected animals (P < 0.001); no difference in survival was observed between the two
control groups (P = 0.139). Specifically, vaccination
with the Env-encoding vector fully protected 8 of 18 mice (44%)
against challenge with MBL-2 leukemia cells and prolonged the survival
time of 3 other vaccinated animals (17%).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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In the present study, we demonstrate that DNA immunization with an M-MSV/M-MuLV Gag-expressing plasmid that included the naturally occurring immunodominant epitope conferred complete protection against subsequent challenge with tumor cells bearing the relevant virus antigens; substantial therapeutic efficacy was also achieved by immunizing mice with a plasmid containing the env gene, which expresses a subdominant epitope that is weakly immunogenic during virus infection.
Our data extend the results of studies by Chen et al. (10)
conducted with B6 mice that demonstrated that the majority of CTL
produced in response to immunization with an F-MuLV-induced leukemia
cell line were specific for a Gag epitope (Gag85-93; CCLCLTVFL). Our findings indicate that Gag85-93 also
represents the immunodominant epitope during the course of the natural
immune response to M-MSV/M-MuLV infection in mice having the
H-2b haplotype. Analyses of tumor regressor mice
showed that (i) the frequency of CTLp against MBL-2 leukemia cells
almost completely paralleled that against EL4 leukemia
cells pulsed with Gag85-93 nonapeptide, (ii) the majority
of the CTL clones from virus-injected mice were specifically directed
against target cells loaded with this peptide, and (iii) the cytotoxic
activity of bulk MLTC against MBL-2 target cells was inhibited in a
dose-dependent manner in the presence of Gag85-93-pulsed EL-4 cells used as cold targets. On the contrary, in the
F-MuLV model, mice infected with the retrovirus generated a CTL
response directed predominantly against a determinant(s) in the F-MuLV
envelope protein (21).
Moreover, we observed that immunization with the Gag-expressing plasmid brought about a striking induction of CTL that were specific for the immunodominant peptide recognized by CTL from tumor regressor mice and, more importantly, resulted in full protection against tumor challenge. The therapeutic effects we obtained are particularly relevant in light of a recent report showing that immunization of B6 mice with the Gag85-93 peptide alone protected only 20 to 40% of animals against tumor development and that the association of this CTL epitope with a virus-specific Env-derived T-helper epitope was needed to achieve nearly complete protection (27).
Two hypotheses may explain the higher protective efficacy of DNA vaccination than of vaccination with a synthetic Gag peptide. It was recently reported that CTL induction requires the participation of dendritic cells that are activated and "licensed" by CD4+ T cells (1, 29, 31). The fact that plasmid DNA is endowed with immunostimulatory properties capable of inducing IL-12 production (24) and dendritic-cell maturation (33) leads to the suggestion that it is able per se to provide dendritic cells with the necessary signal to properly present the gag-derived epitope to CTL.
However, the observation by Ossendorp et al. (27) that administration of the immunodominant Gag peptide in combination with a non-virus-specific T-helper epitope failed to confer protection argues against the hypothesis that bystander help favors the induction of CTL against M-MSV/M-MuLV tumors.
Alternatively, the gag sequence itself might contain a T-helper epitope(s). In this regard, it is noteworthy that a protective epitope capable of activating CD4+ T-helper cells was identified by Miyazawa et al. (26) following immunization with recombinant vaccinia virus expressing the F-MuLV gag gene. This epitope, whose precise sequence has not yet been identified, is located in the N-terminal region of the Gag precursor and presents strong homology to the M-MuLV sequence. Given that DNA vaccination has also been reported to induce the sensitization of CD4+ Th1 cells (7), it is quite likely that immunization with the Gag-expressing plasmid activates specific CD4+ cells which are required for optimal CTL induction and protection against tumor challenge. In fact, we previously observed that in vivo inactivation of CD4+ cell function by treatment with anti-CD4 MAb leads to progressive M-MSV/M-MuLV-induced tumor growth due to the lack of tumor-specific CTL generation (2); accordingly, recent experiments have shown that anti-CD4 MAb treatment during pcDNA3-gag immunization counteracts protection (data not shown).
Previous studies of the M-MuLV system identified a peptide spanning amino acids 189 to 196 of the Env precursor that displays enhanced recognition in B6.C-H-2bm13 mice but behaves as an immunorecessive epitope in B6 mice (32). In agreement with these findings, we observed weak cytotoxic activity against 293Kb cells loaded with this octapeptide in MLTC obtained from tumor regressor mice (Fig. 5). Moreover, only a few CTL clones from tumor regressor mice were able to recognize target cells pulsed with Env189-196 peptide. In contrast, most of the mice that were vaccinated with the Env-expressing plasmid generated CTL that not only recognized Env189-196-pulsed target cells but also efficiently lysed MBL-2 leukemia cells.
Notwithstanding, protection against MBL-2 tumor cell challenge was achieved in less than 50% of the mice vaccinated with pCMV-ecoenv-bpA, thus indicating the existence of a hierarchy in the capacity of different antigenic epitopes to induce a therapeutic immune response against viruses and possibly tumors. The observed limited protection afforded by the Env-encoding plasmid is in line with the results of a study by Ossendorp et al., which showed that the Env189-196 peptide was less effective than the Gag85-93 peptide in inducing protection in experiments involving these peptides combined with the virus-specific helper epitope (27).
The present findings lead to hypotheses regarding the development of an effective DNA-based vaccine against retroviruses or tumors. The possibility of DNA immunization to induce a potentially protective immune response against recessive epitopes might be very advantageous when the immunodominant epitope is lost or the presenting class I molecule is down-regulated. Furthermore, DNA vaccination allows the expression of selected subunits of the infectious agent, thus bypassing the potential risks associated with the use of attenuated viruses, such as reversion to virulence and recombination with endogenous retroviral sequences resulting in the emergence of new infectious viruses.
DNA-based immunization also appears to be more straightforward than epitope-focused immunization procedures (17), such as those involving synthetic peptides, given that DNA vaccination does not require prior identification of the antigenic epitopes and allows the MHC haplotype of the recipient organism to determine which protein will present the immunodominant peptides to effector cells of the immune system.
The encouraging results achieved in DNA-immunized chimpanzees challenged with high human immunodeficiency virus doses (4), as well as very recent reports showing the induction of humoral and cell-mediated immune responses in humans immunized with plasmids encoding human immunodeficiency virus proteins (6, 25), provide tangible evidence that DNA immunization represents a viable approach for vaccination of humans against retroviruses.
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ACKNOWLEDGMENTS |
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We thank S. Mandruzzato, G. Biasi, and B. Macino for critical reading of manuscript; V. Barbieri for technical assistance; and D. M. D'Agostino for editing the manuscript.
This study was supported by grants from the Italian Association for Cancer Research (AIRC), ISS Italy-USA Program on "Therapy of Tumors," Italian Ministry of Public Education. G. Milan is supported by a fellowship from the Italian Foundation for Cancer Research (FIRC).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, I-35128 Padua, Italy. Phone: 39-49-8071859. Fax: 39-49-8072854. E-mail: arosato{at}ux1.unipd.it.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Journal of Virology, March 1999, p. 2280-2287, Vol. 73, No. 3
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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