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Journal of Virology, October 2000, p. 9083-9089, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Induction of CD8 T Cells by Vaccination with Recombinant
Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces
Tumor Growth
Dai-Wei
Liu,1,2
Yeou-Ping
Tsao,1,3
Chang-Hsun
Hsieh,1
Jer-Tsong
Hsieh,4
John T.
Kung,5
Chia-Lien
Chiang,1
Shyh-Jer
Huang,1 and
Show-Li
Chen1,*
Department of Microbiology and
Immunology1 and The Graduate Institute
of Medical Science,2 National Defense Medical
Center, and Institute of Molecular Biology, Academia
Sinica,5 Taipei, and Department of
Ophthalmology, Chang Gung Memorial Hospital and Chang Gung University,
Taoyuan,3 Taiwan, Republic of China, and
Department of Urology, The Southwestern Medical School, Dallas,
Texas4
Received 2 May 2000/Accepted 14 July 2000
 |
ABSTRACT |
The potential of the E5 protein as a tumor vaccine candidate has
not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the
growth of tumors which contain E5 gene expression. Moreover, the E5
vaccine-induced tumor protection occurs through CD8 T cells but not
through CD4 T cells in in vitro assays. In addition, our studies using
knockout mice with distinct T-cell deficiencies confirm that cytotoxic
T-lymphocyte-induced tumor protection is CD8 dependent but CD4
independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.
 |
INTRODUCTION |
Cervical cancer is one of the
leading cancers in the world. Human papillomavirus type 16 (HPV-16) is
the predominant type of virus identified in cervical cancers. It
carries three transforming oncogenes
E5, E6, and E7 (24,
65). Thus, they are unique tumor antigens and serve as ideal
materials for a tumor vaccine. Because E6 and E7 oncoproteins are
consistently retained and expressed, these two oncogenes are attractive
targets for T-cell-based immunotherapy of cervical cancer. Previous
studies have used different modes of E6 and/or E7 immunization to both
experimental and natural papillomavirus-associated tumors, such as (a)
recombinant vaccinia viruses (1, 3, 20, 35, 42, 63), (b)
recombinant adeno-associated virus (36), (c) syngeneic cells
(6, 7), (d) dendritic cells (11, 53), (e) a
cytotoxic T-lymphocyte (CTL) epitope peptide with incomplete Freund's
adjuvant (13), (f) papillomavirus-like particles (22,
37, 49), and (g) Salmonella enterica serovar
Typhimurium (38). These studies demonstrate that cytotoxic T
lymphocytes (CTLs) are the most effective immunological effectors to
protect against tumor growth.
E5 protein is located at the cell surface and reduces cell gap-gap
junction communication (10, 25, 45, 51). E5 is expressed in
earlier stages of neoplastic transformation of the cervical epithelium
during viral infection. Since early lesions usually contain fewer tumor
cells, the immune response may have a higher chance of eradicating
tumor cells in premalignant lesions than in invasive cervical cancers.
In addition, condylomata are reported to have very low levels of major
histocompatibility class I (MHC-I) and MHC-II mRNA; such a lack
probably hampers keratinocyte presentation of antigen, leading to a
decrease in immunological surveillance (41). Recent reports
demonstrated that the lymphocyte proliferation responses to HPV-16 E5
are inversely proportional to the severity of the squamous
intraepithelial neoplasia lesions (SILs) (21). Hence, in
premalignant lesions, including SILs and condylomata, when E5 is still
expressed, using E5 as a vaccine to target E5-expressing cells may be a
good strategy to prevent premalignant lesions from progressing into
invasive cervical cancers. However, the potential of E5 protein as a
tumor vaccine candidate has not been identified. Hence, in this study,
we evaluated the HPV-16 E5 protein delivered by an adenovirus vector as
a tumor vaccine for cervical lesions. The results demonstrate that a
single intramuscular injection of the recombinant adenovirus (rAd)
encoding HPV-16 E5 into syngeneic animals can reduce tumor growth in
lesions which contain E5 gene expression. It is also shown that E5
vaccine-induced protection against tumors is through CD8 T cells but
not through CD4 T cells.
 |
MATERIALS AND METHODS |
Construction of recombinant adenovirus vector containing HPV-16
E5 gene.
To generate replication-deficient recombinant viruses
carrying the HPV-16 E5 gene, we isolated a 0.3-kb BamHI
fragment from HPV-16 E5/pCEP4 which contained the influenza virus
hemagglutinin 1 (HA1) epitope tagged at the 5' terminal end of the
HPV-16 E5 gene and ligated it with pAdE1CMV/pA (30), and it
was named pXCMVHA16E5 (Fig. 1A). The HA1
epitope tag is an 11-amino-acid sequence
(Met-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser) from an HA epitope of
influenza virus, against which a highly reactive monoclonal antibody
was raised. After restriction enzyme mapping, a plasmid containing the
E5 gene was cotransfected with pJM17 into 293 cells to generate
recombinant viruses (30). The genomic structure of the
recombinant adenoviruses containing the E5 gene was confirmed by PCR.
For large-scale virus production, the recombinant viruses were
harvested from 20 plates of 293 cells grown on a P-150 dish after
36 h of infection and subjected to two cycles of CsCl gradient
ultracentrifugation (31). After overnight dialysis, the
stock of viruses was aliquoted and stored at
80°C until use. The
average titers of viral stocks were determined by a plaque assay in
triplicate.

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FIG. 1.
Construction and generation of recombinant adenovirus
encoding HPV-16 E5. (A) The plasmid pXCMVHA16E5 was the recombinant
adenovirus vector containing the HPV-16 E5 gene which had a tagged HA1
epitope at the 5' end (see Materials and Methods). (B) The expression
of the E5 protein by cells transduced with the recombinant adenovirus
rAd-E5. 293 cells were infected at a multiplicity of infection of 25 with rAd-lacZ (lane 1) and rAd-E5 (lane 2). Forty-eight hours later,
cellular proteins were extracted and immunoprecipitated with the HA1
antibody. Then, they were electrophoresed by SDS-PAGE and analyzed with
the HA1 antibody by using Western blotting.
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Cells.
TC-1 is an E6- and E7-expressing tumorigenic cell
line which came from primary lung epithelial cells of C57BL/6 mice
immortalized by HPV-16 E6 and E7 and then transformed with an activated
ras oncogene (57). To establish a C57BL/6
syngeneic mouse tumor model containing the E5 gene, TC-1 cells were
transfected using a liposome method with the HPV-16E5/pHOOK plasmid,
which contained the HPV-16 E5 gene with a 5'-tagged HA1 epitope in the
plasmid vector pHOOKs (Invitrogen, Carlsbad, Calif.). The E5 gene
expression was driven by a minimal human cytomegalovirus (hCMV) early
promoter. Three or 4 weeks following transfection, at least 80 to 100 zeocin-resistant colonies were selected, pooled, and named TC-1/E5.
TC-1 and TC-1/E5 cells were maintained in RPMI 1640 medium supplemented
with 10% fetal calf serum, penicillin-streptomycin (50 U/ml),
L-glutamine (2 mM), sodium pyruvate (1 mM), nonessential
amino acids (2 mM), G418 (0.4 mg/ml), and hygromycin (0.2 mg/ml). They
were grown at 37°C in a 5% CO2 atmosphere.
Animals.
C57BL/6 (H-2b) mice were obtained from
the National Laboratory Animal Breed and Research Center (Taipei,
Taiwan) and maintained in our institute under specific-pathogen-free
conditions. The mice were used at 7 to 10 weeks of age. Knockout I (KO
I) mice are
2-microglobulin (
2m)
/
mutants which
fail to express MHC-I molecules on the cell surface and thus are
virtually devoid of functional CD8
/
T cells (32, 64).
KO II mice are H-2-IA
/
mutants which lack
surface-expressed MHC-II molecules (23) and hence are
virtually devoid of functional CD4
/
T lymphocytes.
Northern blot analysis.
Total cytoplasmic RNA was prepared
from cells and analyzed by Northern blot hybridization. Filters were
washed to remove nonspecifically bound probes, air dried, and then
exposed to Kodak XAR film with Dupont Lightning-plus intensification screens.
Analysis of E5 protein expression.
Cell extracts were
prepared as described previously (9). An antibody to the HA1
epitope (ATCC 12CA5) was added to the extracts, which were then
incubated for 2 h at 4°C. After rotation at 4°C for 2 h,
50 µl of a 1:1 suspension of protein A-Sepharose beads (Pharmacia) in
TBS-BSA (10 mM Tris-HCl pH 7.4; 165 mM NaCl; 10% [wt/vol] bovine
serum albumin) was added, and the mixture was rotated again for 45 min
at 4°C. The beads were pelleted and washed five times with cold
radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl
sulfate [SDS]) plus protease inhibitors (phenylmethylsulfonyl fluoride and leupeptin) and resuspended in 75 µl of sample buffer with
-mercaptoethanol. Samples were heated to 100°C for 4 min and
analyzed by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE). Gels were subjected to immunoblot analysis with HA1 mouse monoclonal antibodies (1:500 dilution, 100 µg/ml) and visualized by an enhanced chemiluminescence system (Amersham) using procedures recommended by the manufacturer.
In vivo tumor prevention and elimination experiments.
To
assay tumor prevention experiments, 5 × 1010 PFU of
rAd-E5 and rAd-lacZ (namely, the Escherichia coli lacZ gene
that is used to monitor
-galactosidase [
-Gal]) or
phosphate-buffered saline (PBS) was injected into 10 mice from each
group. After 1 week, 5 × 104 TC-1/E5, or TC-1 tumor
cells, were injected subcutaneously (s.c.) into the left flank of
C57BL/6 mice. The mice were then monitored once a week for tumor
growth. To assay tumor elimination, 5 × 104 TC-1/E5,
or TC-1 tumor cells, were injected s.c. into the left flank of C57BL/6
mice. After 1 week, the mice were vaccinated with 5 × 1010 PFU of either rAd-E5, rAd-lacZ, or PBS (mock). They
were injected intramuscularly (i.m.) into the hind leg tibialis
anterior muscles. The mice were then monitored once a week for tumor growth.
Cell-mediated lymphocyte cytotoxicity.
Cell-mediated
cytotoxicity was measured using a 51Cr release assay and
performed using standard protocols (57). Splenocytes were
harvested from mice that were vaccinated with rAd-E5, rAd-lacZ, or PBS
2 weeks previously. The splenocytes were cocultured with mitomycin-treated TC-1/E5 cells or a combination of E5 peptides which
cover the whole E5 protein (stimulators) (5, 21, 29) for 6 days. Various numbers of stimulator-treated splenocytes (effector) were
added to 104
Na251CrO4-labeled target cells
(TC-1/E5) in 100 µl of culture medium in 96-well U-bottom plates.
After a 4-h incubation at 37°C, 25 µl of culture supernatant was
collected for gamma radiation counting. To characterize the roles of
CD4 and CD8 T lymphocytes in E5-induced cytotoxicity the anti-CD4
monoclonal antibody (GK1.5) or anti-CD8 monoclonal antibody (2.43) was
mixed with effector cells, respectively, before being added to target
cells in a final concentration of 50 µg/ml to block CD4+
or CD8+ T lymphocytes. The mean percentage of specific
lysis of triplicate wells was calculated as follows, where cpm is
counts per minute: % specific lysis = {[cpm of experimental
release
cpm of spontaneous release]/[cpm of maximum (1%
Triton X-100) release
cpm spontaneous release]} × 100%.
 |
RESULTS |
Expression of HPV-16 E5 protein in the adenovirus-transduced E5
gene.
The plasmid pXCMVHA16E5, which carries the HPV-16 E5 gene,
was constructed by inserting the E5 gene into the adenovirus vector pAdE1CMV/pA (Fig. 1A). The replication-defective recombinant
adenoviruses (rAd-E5) were generated as described in Materials and
Methods. In rAd-E5, E5 gene expression was driven by a minimal hCMV
early promoter. To detect the expression of the E5 protein, 293 cells were infected at a multiplicity of infection of 25 with either rAd-E5
(Fig. 1B, lane 2) or rAd-lacZ (Fig. 1B, lane 1). Forty-eight hours
after infection, total cellular proteins were extracted and
immunoprecipitation assay or Western blot analysis was performed. Figure 1B shows that only rAd-E5-infected cells could express the
12.5-kDa E5 protein.
Establishment of E5 syngeneic cells.
We have established
C57BL/6 syngeneic cells containing the E5 gene (TC-1/E5) as described
in Materials and Methods. E5 gene expression in TC-1/E5 was monitored
by Northern blot analysis and immunoprecipitation or Western blot
analysis. Figures 2A and B, lane 1, show
E5 RNA and E5 protein in TC-1/E5 cells, respectively, but not in TC-1/V
cells (lane 2, containing the vector only).

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FIG. 2.
Establishment of C57BL/6 syngeneic cells containing the
E5 gene. TC-1/E5 cells were generated by transfecting the plasmid
HPV-16E5/pHOOK into TC-1 cells (see Materials and Methods). (A)
Northern blot analysis identified the E5 RNA expression in TC-1/E5
cells (lane 1), but not in TC-1/V cells (lane 2). A
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to
ensure equal RNA loading. (B) E5 protein expression was found in
TC-1/E5 cells (lane 1), but not in TC-1/V cells (lane 2), by
immunoprecipitation and Western blot assay with the HA1 antibody.
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Vaccination with rAd-E5 generates tumor prevention and protection
functions against challenge with TC-1/E5 tumor cells.
To assess
the degree of prevention of tumor cell growth, 10 C57BL/6 mice of each
group were vaccinated with 5 × 1010 PFU of either
rAd-E5, rAd-lacZ, or PBS (mock) i.m. One week after vaccination, the
mice were injected s.c. with 5 × 104 TC-1/E5 or TC-1
tumor cells. The tumor volume was measured once a week. As shown in
Fig. 3, vaccination with rAd-E5
significantly retarded TC-1/E5 cell-induced tumor development while
inoculation of rAd-lacZ or PBS had no effect, but it could not prevent
TC-1 cell-induced tumor growth.

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FIG. 3.
In vivo tumor prevention assay. Groups of 10 mice were
immunized with 5 × 1010 PFU of rAd-E5, rAd-lacZ, or
PBS (mock). One week later, the immunized mice were injected s.c. with
5 × 104 TC-1/E5 or TC-1 tumor cells. Then, the tumor
volume was monitored once a week. The data are the means and standard
errors of each group.
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To evaluate the tumor treatment effect of the rAd-E5 vaccination, 10 C57BL/6 mice of each group were injected s.c. with 5
× 10
4 TC-1/E5 cells. One week following the tumor cell
injection, they
were immunized i.m. with 5 × 10
10 PFU
of either rAd-E5, rAd-lacZ, or PBS (mock). Then, the tumor
volume was
measured once a week. As shown in Fig.
4,
vaccination
with rAd-E5 significantly eliminated TC-1/E5 cell-induced
tumor
growth while inoculation of rAd-lacZ or PBS had no effect, but
it
could not reduce TC-1 cell-induced tumor growth.

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FIG. 4.
In vivo tumor elimination assay. Groups of 10 mice were
injected s.c. with 5 × 104 TC-1/E5 or TC-1 tumor
cells and, after 1 week, were immunized with 5 × 1010
PFU of rAd-E5, rAd-lacZ, or PBS (mock). The tumor volume was monitored
once a week. The data are the means and standard errors of each
group.
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From the data of Fig.
3 and
4, we observed small-volume tumors in the
E5-vaccinated mice which might express no or low E5.
This may be the
reason that E5-specific CTLs cannot eradicate
them. In the future, we
will look into the differential gene expression
of E5 in the tumor and
investigate the cytolytic effects by
vaccination.
Cellular immune response in mice immunized with rAd-E5.
To
elucidate the mechanism of protection against TC-1/E5 tumors, we
determined whether a CTL response was induced in rAd-E5-immunized mice.
Spleen cells from C57BL/6 mice immunized with either rAd-E5, rAd-lacZ,
or PBS were isolated and stimulated in vitro with mitomycin-treated TC-1/E5 cells (Fig. 5C and D) or the
combination of E5 peptides which cover the whole E5 protein (Fig. 5A
and B) (5, 21, 29). These stimulated spleen cells were then
tested for recognition and lysis of 51Cr-labeled target
cells, including the TC-1/E5 tumor cells expressing the E5 gene (Fig.
5A and C) and B16F1, which was a syngeneic C57BL/6 cell line lacking E5
gene expression (Fig. 5B and D). As shown in Fig. 5, spleen cells from
rAd-E5-immunized animals had CTL activity to lyse TC-1/E5 target cells
(Fig. 5A and C) but not B16F1 cells (Fig. 5B and D). Cells from
rAd-lacZ- or mock-immunized mice had no effect. In addition, since the
HA1 epitope tagged the 5' end of the E5 gene, we also assayed CTL
activity by using the HA1 peptide as a stimulator in E5-vaccinated mice
to rule out the possibility that the response was induced by HA1
instead of E5. Figure 5E and F show that HA1-specific T cells could not lyse TC-1/E5 and B16F1 cells, respectively. Taken together, it is
evident that rAd-E5 vaccine-induced tumor protection is through E5-specific CTL cells.

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FIG. 5.
HPV-16 E5-specific CTL responses induced by immunization
with rAd-E5. C57BL/6 mice were i.m. injected with rAd-E5. Two weeks
after the vaccination, their spleens were collected and analyzed by an
in vitro CTL assay. Target cells were TC-1/E5 cells (A, C, and E) and
B16F1 cells (B, D, and F). The stimulators were as follows: a
combination of E5 peptides which cover the whole E5 protein (A and B),
mitomycin-treated TC-1/E5 cells (C and D), and a synthetic HA1 peptide
(Met-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser) (E and F). The data are
the averages of data from five vaccinated mice.
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CD8-dependent immunity on tumor protection by vaccination with
rAd-E5.
To understand the relative roles of CD4 and CD8 T cells in
rAd-E5 vaccine-induced tumor protection, mice deficient in CD4 and CD8
T cells as a result of targeted gene disruption at
2m and
MHC-II, respectively, were studied. The sources of CD8 and CD4 T-cell-deficient mice were
2m
/
and
MHC-II
/
mice on a C57BL/6 background, respectively
(23, 32, 64).
2m
/
and
MHC-II
/
mice were kindly provided by B. J. Fowlkes
(National Institutes of Health, Bethesda, Md.) and were bred under
specific-pathogen-free conditions. Groups (n = 6) of
CD4 (KO II) and CD8 (KO I) T-cell-deficient mice were injected with
5 × 104 TC-1/E5 cells, followed by vaccination with
either rAd-E5 or control rAd-lacZ 1 week later. Figure
6 shows evident tumor growth in CD8
T-cell-deficient groups, but not in CD4 T-cell-deficient mice.
Furthermore, we blocked CD8 T cells or CD4 T cells by coculturing effector cells with anti-CD8 or anti-CD4 antibody, respectively, and
assayed the in vitro CTL response by rAd-E5-immunized mice. As shown in
Fig. 7, the lysis of E5-stimulated
splenocytes (effector cells) to target cells (TC-1/E5) significantly
dropped when effector cells were cocultured with anti-CD8 antibody, but
not with anti-CD4 antibody or PBS (mock). Taken together, these data
suggest that CD8 T cells, but not CD4 cells, participate in rAd-E5
vaccine-induced tumor reduction.

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FIG. 6.
CD8-dependent lymphocytes for tumor eradication by
rAd-E5 vaccination. KO I (CD8-deficient) and KO II (CD4-deficient) mice
were s.c. injected with 5 × 104 TC-1/E5 tumor cells.
One week later, they were divided into two groups for treatment with
5 × 1010 PFU of rAd-E5 or rAd-lacZ. Each group
consisted of six mice. The tumor volume was monitored once a week.
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FIG. 7.
CTL response is CD8 dependent and CD4 independent.
C57BL/6 mice were i.m. injected with rAd-E5. Two weeks after the
vaccination, the splenocytes were collected and cocultured with
anti-CD4 antibody, anti-CD8 antibody, or PBS and then were analyzed by
an in vitro CTL assay. Target cells and stimulators were TC-1/E5 cells
and mitomycin-treated TC-1/E5 cells, respectively. The data are from
three independent experiments.
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 |
DISCUSSION |
This is the first demonstration that HPV-16 E5 can be regarded as
a tumor vaccine to suppress tumor growth. Previous studies have
reported that recombinant vaccinia virus expressing the E5 gene of
bovine papillomavirus type 1 (BPV-1) can immunize against BPV-1 tumor
cells (43), but vaccination with the recombinant vaccinia
virus expressing the HPV-16 E5 protein fails to influence tumor
development (42). Such a failure to eradicate tumors by using a vaccinia virus delivery system may be due to the fact that they
cannot detect the E5 gene expression in tumor cells, or perhaps the
vaccinia virus, unlike the adenovirus, cannot assist the E5 protein to
enter the MHC-I or -II pathway for antigen presentation. However, our
study manifests that vaccination with rAd-E5 can reduce the growth of
tumors via CTL activity. While investigating the roles of CD4 and CD8 T
lymphocytes in rAd-E5 vaccine-induced tumor protection, we found that
CD8 knockout mice vaccinated with rAd-E5 lost tumor-reducing activity,
but CD4 knockout mice did not lose tumor-reducing activity (Fig. 6).
This was further confirmed by an in vitro E5-specific CTL assay using
incubation with anti-CD4 or anti-CD8 antibody to block CD4 or CD8 cell
function (Fig. 7). Our observation means that CTL activity is caused
only by CD8 T cells activated by vaccination with the rAd-E5, and not
by CD4 T cells.
In this study, we demonstrated that E5 vaccine delivered by adenovirus
vectors can induce tumor reduction. The potential for tumor vaccine
development using adenovirus vectors has been explored widely. Previous
studies have shown that mice vaccinated with a recombinant adenovirus
encoding the tumor-specific antigen p815A present on mouse mastocytomas
can induce an anti-p815A CTL response (55) and
eradicate tumors. A recombinant adenovirus encoding
-Gal,
administered with exogenous interleukin-2 (IL-2), can lead to a
reduction of an established
-Gal-expressing CT26 murine colorectal
cancer (8). Similarly, immunization with a recombinant adenovirus encoding the melanoma-associated antigen (gp100) can protect
mice from intradermal challenge with murine B16 melanoma cells
via CD8 T cells (61). In addition, an adenovirus vector as a
vaccine against virus challenges has also been developed. For
example, cattle immunized with a recombinant adenovirus encoding the
structural proteins of the foot-and-mouth disease virus can produce
significant protection against viral challenge (48). In
mice, protection has been demonstrated against subsequent challenge by
a variety of viruses by prior immunization with an appropriate recombinant adenovirus-mediated viral gene expression. Examples of such
viruses include rabies virus (46), tick-borne encephalitis virus (27), rotavirus (2), herpes simplex virus
(19), murine hepatitis virus (56), measles virus
(15, 16), and simian immunodeficiency virus (SIV)
(14). All these studies demonstrate that an adenovirus
vector can help a transgene elicit a CTL response in mice against
antigen-specific tumors (8, 55, 61) and induce both humoral
and cellular immunity against subsequent virus challenges (2,
14-16, 19, 27, 46, 56).
In this study, we chose a single injection of rAd-E5 for vaccine
delivery. Recombinant adenoviruses are efficient carriers for
vaccination, as described above (26, 62). It is usually not
efficient to reintroduce an adenovirus vector for a booster response.
This is mainly due to the adenovirus-induced neutralizing antibodies
which are directed against the fiber and hexon of adenovirus in
infected mice (12, 59), rats (39), cotton rats
(60), and rhesus monkeys (28) and which can
particularly affect secondary entry and delivery of the vector. But, no
adenovirus immunity to transgene expression has been reported. However,
one recent report showed that preexisting immunity to the adenovirus
does not prevent antitumor protection following intratumoral
administration of an IL-12-expressing adenovirus vector (4).
Thus, the influence of immunogenicity from the adenovirus on vaccine
efficacy is still mysterious. But if humoral immune responses reveal
certain limitations of the adenovirus vectors that may affect its
potency and readministration for gene therapy of cancer, then a single
immunization may overcome this booster effect, in which a neutralizing
anti-adenovirus antibody abolishes the vector-directed gene expression
(16, 18).
The importance of HPV as a necessary but insufficient component in the
development of cervical cancers has been well established (24,
65). Numerous cofactors can explain the imbalance between the
very high prevalence of HPV infection and the relatively low incidence
of anogenital cancers in the United States (17, 44). The
high prevalence of HPV-associated SILs in human immunodeficiency virus
(HIV)-infected individuals implies that the host immune response may
play a significant role in the development of HPV-associated cancers
(40, 52). The higher rates of HPV infection and SILs in
HIV-infected women are thought to be attributed specifically to a
decrease in CD4 T cells that causes the immune system to be impaired
(33, 40, 47, 52, 54). HIV infection adversely affects the
synthesis of Th1 cytokines by CD4 T cells, but not gamma interferon
(INF-
) synthesis by CD8 T cells of women with active HPV infection
(34). The increase in IFN-
+ CD8 T cells is a
phenotype consistent with CTLs. These unaffected INF-
+
CD8 T cells are less likely to be HPV specific as there is a higher
incidence of HPV-related cervical SIL for HIV-positive, HPV-positive
women than for HIV-negative, HPV-positive women (34). In
this study, we demonstrated that the E5 vaccine-induced CTL response is
CD8 dependent but CD4 independent. Accordingly, HIV patients with
higher HPV loads have CD8 T-cell counts similar to those of healthy
women but lack CD4 T cells. Thereafter, E5 as a therapeutic
vaccine may have the capacity to stimulate CD8 cells into
E5-specific CTLs to eradicate E5-expressing dysplasia cells;
thus, it may have a higher chance of preventing SILs progressing into invasive cervical cancers in both HPV infection alone and HPV-HIV infection.
In summary, our study demonstrates that a single i.m. injection of
recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic
animals could reduce tumor growth. It also shows that the E5
vaccine-induced tumor protection is through a CD8-dependent and
CD4-independent CTL response. Hence, HPV-16 E5 can be regarded as a
tumor rejection antigen.
 |
ACKNOWLEDGMENTS |
We are grateful to T. C. Wu for providing TC-1 cells,
B. J. Fowlkes for providing
2m
/
and
MHC-II
/
mice, and Judy Perry for proofreading the manuscript.
This work was supported by National Science Council grant NSC
87-2312-B106-003.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China. Phone: 886-2-87923100, ext. 18543. Fax:
886-2-87924885. E-mail: yptsao{at}kimo.com.tw.
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Journal of Virology, October 2000, p. 9083-9089, Vol. 74, No. 19
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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