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Journal of Virology, September 1999, p. 7877-7881, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Papillomavirus Type 16 E7 DNA Vaccine: Mutation in the Open
Reading Frame of E7 Enhances Specific Cytotoxic T-Lymphocyte
Induction and Antitumor Activity
Wei
Shi,1
Ping
Bu,1
Jianzhong
Liu,1
Axel
Polack,2
Susan
Fisher,3 and
Liang
Qiao1,*
Department of Microbiology and
Immunology1 and Cardinal Bernardin
Cancer Center,3 Stritch School of Medicine,
Loyola University Chicago, Maywood, Illinois 60153, and
National Research Center for Environment and Health,
Institute of Clinical Molecular Biology and Tumor Genetics, D-81377
Munich, Germany2
Received 11 January 1999/Accepted 25 May 1999
 |
ABSTRACT |
A human papillomavirus type 16 E7 DNA vaccine with the open reading
frame encoding mutations in two zinc-binding motifs expressed a rapidly
degraded E7 protein. This vaccine induced a significantly stronger
E7-specific cytotoxic T-lymphocyte response and better tumor protection
in mice than did a wild-type E7 DNA vaccine expressing a stable E7 protein.
| |
TEXT |
Human papillomavirus (HPV)
genes and their products have been identified in most anogenital
cancers, and HPV type 16 (HPV 16) is the most common one associated
with severe cervical dysplasia and cancers (25, 45).
Certain early viral genes, such as HPV 16 E6 and E7, are
expressed constantly in cervical cancer cells (13, 35).
Immunogenicity of HPV 16 E7 has been demonstrated by using overlapping
peptides spanning the full length of E7, by recombinant E7 proteins, by
recombinant vaccinia virus containing the E7 open reading frame (ORF),
or by CD80+ and HPV 16 E7+ tumor cells (1,
6, 8-10, 15-18, 20, 21, 23, 27, 34, 37, 38). Certain mouse and
human T-cell epitopes of the E7 protein have been identified (1,
10, 15, 21, 31, 37, 38).
DNA-based immunization can induce host immune responses (2, 4, 19,
32, 41). DNA vaccination is accomplished by the expression of
inoculated DNA encoding the gene of interest, with a mammalian promoter
or enhancer and other DNA sequences that enable the gene to be
expressed within mammalian cells (11, 12, 26, 43). To
prevent viral infections and to treat viral diseases, cytotoxic T
lymphocytes (CTL) are crucial. DNA immunization can potentially be very
effective at inducing a major histocompatibility complex (MHC) class
I-restricted CTL response because DNA encoding the antigen of interest
is delivered directly into the cell, where its gene product accesses
the MHC class I antigen presentation pathway. DNA vaccines have the
following advantages over other vaccines. (i) The vaccines can be
prepared easily. Genes inserted into the vector (plasmid) can be
modified easily, allowing convenient removal or insertion of certain
sequences. (ii) DNA vaccines are temperature stable, which allows
economical transportation, especially in underdeveloped countries.
(iii) The immune responses induced by DNA vaccines are long
lasting. (iv) DNA immunization will not induce an immune response
against the DNA vector itself, so the DNA vaccine can be used
repeatedly. This feature is important because treatment of cancer may
require repeated immunization, and an immune response against the
vector might reduce the efficacy of the vaccine. Although DNA vaccines
have been shown to induce CTL responses, not all immunized individuals
develop CTL (7, 30, 33, 44). Therefore the potency of DNA
vaccines in inducing CTL must be improved.
Because cervical cancers express the E7 antigen, we explored whether a
DNA vaccine can be used to induce an E7-specific CTL response
and protect against tumor challenge. However, experimental evidence demonstrates that HPV 16 E7 has transforming potential. HPV 16 E7 has been shown to transform established cells (22, 42).
The HPV 16 E7 ORF is predicted to encode a 98-amino-acid protein that
is phosphorylated, e.g., in the casein kinase II phosphorylation motifs
just downstream of the retinoblastoma protein interaction motif in
conserved region 2 (28, 36). A region within the E7 protein
contains two Cys-X-X-Cys motifs. Mutations affecting only one of the
Cys-X-X-Cys repeats, which are conserved between different HPV E7
proteins, severely reduced the transforming activity but did not
totally destroy it. Double mutants in which both motifs were disrupted
had little transforming activity (14). Further, the motifs
are believed to form a zinc finger and thus may be important in
maintaining the stability of the E7 protein (3, 5). An
unstable protein has greater potential to generate CTL responses than a
stable one (39, 40). To develop a safe DNA vaccine with low
or no transforming activity and to increase the instability of the E7
protein, we produced a plasmid that contains an HPV16 E7 double
mutation in the two Cys-X-X-Cys repeats (58 Cys 
Gly, 91 Cys Gly).
Construction of HPV 16 E7 DNA vaccines.
DNA vaccines used in
our study were the plasmids that contain the HPV 16 wild-type E7 ORF or
double mutant E7 (58 Cys Gly, 91 Cys Gly) ORF under the control
of the cytomegalovirus immediate-early promoter and enhancer. The ORF
encoding the wild-type E7 protein or the E7 double mutant was amplified
by PCR from HPV 16 DNA. For wild-type E7, 5' primer
AGTCGCATGCATCATGCATGGAGAT and 3' primer CCCGCATGCTTATGGTTTCTGAGAAC were used. For the E7
double mutant, overlapping PCR was used. We generated the first mutant
fragment (58 Cys Gly) by using 5' primer
AGTCGCATGCATCATGCATGGAGAT and 3' primer
ACTTGCAACCAAAGGTTAC. The second mutant
fragment (58 Cys Gly, 91 Cys Gly) was generated by using 5'
primer GTAACCTTTGGTTGCAAGT and 3'
primer
CCCGCATGCTTATGGTTTCTGAGAACAGATGGGGCCCAC.
We then performed overlapping PCR to combine two fragments by
using 5' primer AGTCGCATGCATCATGCATGGAGAT and
3' primer
CCCGCATGCTTATGGTTTCTGAGAACAGATGGGGCCCAC. The ORF of E7 or E7 mutant was inserted into the plasmid BC219 SphI site to generate BC219-E7 or BC219-E7 mutant
(29). For large-scale preparations of plasmid DNA,
transformed Escherichia coli DH5
, bacteria were grown in
LB medium in the presence of ampicillin, and plasmids were
extracted by the alkaline lysis method followed by two rounds of
purification on cesium chloride density gradients. DNA
concentrations were determined by measuring the optical density
at 260 nm, and the integrity of the plasmids as well as
the absence of contaminating E. coli DNA or RNA were checked by agarose gel electrophoresis. DNA was stored at
20°C in Tris-EDTA buffer. For injection, DNA was diluted in
phosphate-buffered saline to a final concentration of 2 µg/µl.
Mutant E7 DNA vaccine expresses an unstable E7 protein.
To
determine whether both wild-type E7 and mutant E7 DNA vaccines express
E7 protein, we transfected a cell line (RMA) with the plasmids and
determined expression of the E7 protein by immunoprecipitation.
RMA cells were transfected with each plasmid (BC219-E7 mutant and
BC219-E7, BC219), using a SuperFect transfection kit (Qiagen, Hilden,
Germany) according to the manufacturer's recommendations. At 48 h
after transfection, hygromycin B (final concentration, 600 µg/ml) was
added to the cell culture medium. After cloning by limiting dilution,
the transfectants were maintained in a medium with hygromycin B for 2 weeks.
Stable transfectants (RMA-BC219-E7 mutant,
RMA-BC219-E7, and RMA-BC219) were harvested and washed once
with cold phosphate-buffered
saline. Then 10
6 cells from
each cell line were starved for 30 min in methionine-free
medium, after
which
35S-labeled methionine was added (0.1 mCi/ml, final
activity; Amersham,
Arlington Heights, Ill.). Cells were incubated for
4 h and then
lysed on ice with lysis buffer (1% Nonidet P-40, 150 mM NaCl,
50 mM Tris-HCl [pH 8.0]), and the same amount of cellular
proteins
from each cell line were immunoprecipitated on ice for 2 h with
6 µl of mouse anti-HPV 16 E7 monoclonal antibody (Zymed
Laboratories
Inc., South San Francisco, Calif.). Fifty microliters of
protein
A-agarose beads (Sigma, St. Louis, Mo.) was added to the
antibody-antigen
products, and then the mixture was incubated for
1 h at 4°C with
rocking. Beads were washed three times with the
lysis buffer,
and samples were electrophoresed under reducing
conditions on
sodium dodecyl sulfate-15% polyacrylamide gels. Gels
were dried
and exposed to photographic film. As shown in Fig.
1, the E7 protein
was detected in the
cell line transfected with BC219-E7, whereas
the mutant E7 protein was
barely detectable in the cell line transfected
with BC219-E7
mutant.
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FIG. 1.
Immunoprecipitation of wild-type E7 and mutant E7
proteins. Transfectants (RMA-BC219, RMA-BC219-E7, and RMA-BC219-E7
mutant) were metabolically labeled with [35S]methionine.
For the right-hand set of transfectants, the proteasome inhibitor ALLN
was added at 20 µg/ml 30 min prior to addition of the label and was
maintained at this concentration in all further manipulations. An
immunoprecipitation was carried out with a monoclonal anti-HPV 16 E7
antibody, and the products were separated by electrophoresis. The
absence () or presence (+) of ALLN is indicated.
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To further distinguish failure of synthesis from degradation of E7
protein, we used proteasome inhibitor
N-acetyl-leucyl-norleucinal
(ALLN) in the
immunoprecipitation. Thus, calpain inhibitor I (ALLN),
which is a
cell-permeable synthetic tripeptide with an aldehyde
at its C
terminus and specifically inhibits the activity of cysteine
proteases,
was added to cells at a final concentration of 20 µg/ml
30 min before
addition of radiolabel and was maintained at this
concentration
throughout labeling, harvesting, and immunoprecipitation.
As shown in
Fig.
1, the quantity of E7 protein in cells transfected
with BC219-E7
was not significantly changed in the presence of
the inhibitor,
suggesting that the E7 protein is stable in the
cell. In contrast, in
the presence of ALLN, mutant E7 protein
was detected at levels similar
to those of wild-type E7. Our data
demonstrate that plasmid BC219-E7
mutant can express E7 protein,
but the mutant E7 protein is rapidly
degraded in the
proteasome.
Mutant E7 vaccine induces a stronger CTL response against HPV 16 E7
than wild-type E7 vaccine.
We immunized C57BL/6 mice
(H-2b) intramuscularly with 100 µg of either
BC219-E7, BC219-E7 mutant, or control BC219 plus incomplete Freund's
adjuvant (five mice per group) to determine whether these vaccines
induce an HPV 16 E7-specific CTL response. Six- to 8-week-old female
C57BL/6 mice (purchased from The Jackson Laboratory or Harlan)
were used. All mice were kept under pathogen-free conditions. Two
weeks after the DNA immunization, mice (five per group) were boosted
subcutaneously with 2.5 × 104 HPV 16 E7-expressing
tumor cells (RMA-E7 cells). Four days later, spleen cells were isolated
from each mouse. We boosted mice in vivo with the E7-positive cells to
shorten in vitro stimulation time and generate repeatable results.
After incubation in a nylon wool column for 1 h at 37°C in
5% CO2, T cells were washed through the column with
complete cell culture medium. The cells were cultured at 37°C in 5%
CO2 for 7 days in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), interleukin-2 (10 U/ml), and E7 peptide49-57 (RAHYNIVTF,
H-2Db-restricted epitope; 5 µg/ml). The
CTL specificity was determined in a standard cytotoxicity assay
(24). If the mice had not been primed by the DNA vaccines,
no E7-specific CTL activity was detected at day 4 after the boost. If
the mice had been effectively immunized, E7-specific CTL could be
measured easily at day 4 after the boost. As shown in Fig.
2, lymphocytes isolated from both
BC219-E7 and BC219-E7 mutant groups showed lytic activity against
RMA-E7 cells, whereas lymphocytes isolated from BC219-immunized mice
did not kill RMA-E7 cells. In comparison, immunization with BC219-E7
mutant induced a significantly stronger specific CTL response
against RMA-E7 than BC219-E7. The lymphocytes from all three groups
were unable to kill the control cell line RMA-neo (data not shown).
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FIG. 2.
Spleen lymphocytes from mice immunized with control
vector (BC219) or HPV 16 E7 DNA vaccines (BC219-E7 and BC219-E7 mutant
[mut]) were isolated and stimulated in vitro for 1 week with HPV 16 E7 peptide (amino acids 49 to 57). The specific lysis of the spleen
cells against RMA-E7 cells (target cells) was determined in a standard
6-h 51Cr release assay at different effector-to-target
ratios. Results are the means of specific lysis from five different
mice, and standard deviations were  10%. The CTL response induced by
the mutant E7 vaccine is significantly stronger than the one induced by
the wild-type E7 vaccine (P < 0.01).
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HPV 16 E7 mutant vaccine protected 100% of mice against tumor
challenge.
In the next experiment, we determined whether E7
DNA immunization could induce protective immunity against challenge
with HPV 16 E7-positive tumors. First we developed an E7-positive tumor model by transfecting RMA cells (B6 syngeneic tumor cell line) with
plasmid pCI-neo-E7. A control cell line, RMA-neo, was developed by
transfecting RMA cells with plasmid pCI-neo. The cells were maintained
in RPMI 1640 (GIBCO-BRL, Gaithersburg, Md.) supplemented with 10%
heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and G418 (800 µg/ml; Bio-Rad, Hercules, Calif.).
Mice (10 per group) were immunized intramuscularly with 100 µg of
BC219-E7 mutant, BC219-E7, or BC219 plus incomplete Freund's
adjuvant.
On day 14 after immunization, the mice were challenged
subcutaneously
with 2.5 × 10
4 RMA-E7 tumor cells and monitored every
3 days. This dose of tumor
cells for challenge is the minimal dose to
induce tumors in 100%
of inoculated mice, based on our previous
experiments. Tumor diameter
was measured by two investigators blinded
to the
groups.
On day 13 of challenge, RMA-E7 tumors started growing in four mice
immunized with BC219; by day 22, all of the mice in this
group
developed tumors. Three of the mice immunized with BC219-E7
developed
RMA-E7 tumors on day 15 of tumor challenge, and only
two mice in this
group did not develop tumors by the date of sacrifice.
In contrast,
none of the mice immunized with BC219-E7 mutant developed
an RMA-E7
tumor (Fig.
3). There was a statistically
significant
difference in tumor take between the groups of mice. The
mice
that grew tumors were sacrificed at day 28, and the tumor size
and
weight were assessed. Both tumor size (Fig.
4) and tumor weight
(Fig.
5) of the mice immunized with BC219-E7
were significantly
less than those of the mice immunized with BC219
(
P < 0.01). The
mice immunized with BC219-E7 mutant
did not develop tumors for
at least 6 months. As controls, mice (10 per
group) immunized
with the three different vaccines were challenged with
RMA-neo
cells in the same way. The RMA-neo tumors grew in all three
groups
of mice, and there was no significant difference in tumor take,
tumor size, and tumor weight between the groups (data not shown).
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FIG. 3.
Tumor incidence in mice (10 per group) immunized with
BC219, BC219-E7, or BC219-E7 mutant DNA vaccine. The mice were
challenged with 2.5 × 104 RMA-E7 cells 2 weeks after
the immunization. There were significant differences between each group
(P < 0.001).
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FIG. 4.
Tumor size in mice immunized with BC219, BC219-E7, or
BC219-E7 mutant DNA vaccine. Two weeks after the immunization, mice (10 mice per group) were challenged with 2.5 × 104 RMA-E7
cells. The data represent the size of individual tumors measured at the
end of week 4 after tumor challenge. The median of the size is shown by
bars. The tumor size from the mice immunized with BC219-E7 is
significantly smaller than that from the mice immunized with BC219
(P < 0.01).
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FIG. 5.
Tumor weight in mice immunized with BC219, BC219-E7,
BC219-E7 mutant DNA vaccine. Two weeks after the immunization, mice (10 mice per group) were challenged with 2.5 × 104 RMA-E7
cells. The data represent the weight of individual tumors measured at
the end of week 4 after tumor challenge. The median of the weight is
shown by bars. The tumor weight from the mice immunized with BC219-E7
is significantly less than that from the mice immunized with BC219
(P < 0.01).
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The differences in continuous measures among groups were compared by
analysis of variance. Between-group comparisons were
made with the
Duncan test. Differences in time to tumor take were
examined using
Kaplan-Meier actuarial survival techniques. In
all cases a two-sided
alpha level of 0.05 was considered statistically
significant.
Our data indicate that a DNA vaccine encoding the wild-type E7 protein
can induce an E7-specific CTL response and some tumor
protection.
However, because the E7 protein is known to have transforming
activity,
this DNA vaccine might have oncogenic potential. Although
any
E7-expressing cells should be killed by the CTL induced by
the DNA
vaccine, some plasmids might be retained in MHC class
I-low-expressing
cells that might resist CTL lysis; thus, transformation
of these cells
could occur. Mutation in the E7 protein, which
abrogates the
transforming activity, helps to ensure the safety
of the DNA
vaccine.
Although the wild-type E7 DNA vaccine protected mice against challenge
with HPV 16 E7-positive tumor cells, the protection
was not complete.
There are two possible reasons: (i) RMA-E7 cells
grow relatively fast
and might override the CTL induced by the
E7 DNA vaccine; and (ii) the
number of CTL generated by the vaccine
might be too low. Because the E7
plasmid is believed to be taken
up by antigen-presenting cells, e.g.,
dendritic cells, the E7
protein is likely produced by these cells.
Because E7 cannot be
secreted, it enters the MHC class I pathway
instead of the MHC
class II pathway. Consequently, no E7-specific T
helper cells
are generated, which might limit the expansion of
E7-specific
CTL.
Our data indicate that mutation in the zinc-binding motifs near the C
terminus of E7 leads to rapid degradation of the protein.
This finding
is based on the fact that the mutant E7 can be better
detected in the
presence of a proteasome inhibitor (ALLN). Because
a zinc finger might
be important in maintaining the stability
of the protein, the mutation
in the motifs might abrogate the
zinc-binding activity and result in
instability of the protein.
Our data suggest that the mutant E7 may be
degraded rapidly in
the proteasome and a large amount of antigenic
peptides may be
transported to endoplasmic reticulum. Consequently,
more MHC class
I molecules of antigen-presenting cells present the E7
peptide
(amino acids 49 to 57), resulting in an enhanced CTL response
and tumor protection. Townsend et al. reported that defective
presentation to class I-restricted CTL in vaccinia virus-infected
cells
is overcome by enhanced degradation of influenza virus hemagglutinin
antigen (
40). Tobery and Siliciano demonstrated that rapid
intracellular
degradation of human immunodeficiency virus type 1 Env or
Nef
protein induced an enhanced CTL response in vivo after recombinant
vaccinia virus immunization (
39). Recently it has been shown
that ubiquitination of a viral protein enhances CTL induction
and
antiviral protection in DNA immunization (
33). Thus, those
data together with ours suggest that acceleration of protein
degradation
leads to enhanced antigen presentation in the context of
MHC class
I and subsequently CTL
induction.
The potency of a DNA vaccine for the generation of CTL is relatively
weak. It has been shown that only some individuals immunized
with HIV
Nef, Rev, or Tat DNA vaccines had CTL responses in their
peripheral
blood (
7). It has also been shown that 50 to 75%
of animals
immunized with pCMV-NP DNA vaccines developed CTL (
30,
33,
44). It is predictable that the individuals without a
measurable
CTL response will not have effective protection against
viral
infection. This was also true in a tumor system, as we showed
that the
wild-type E7 DNA vaccine did not protect all mice against
tumor
challenge, although it induced some CTL response specific
for the E7.
Therefore, CTL responses can be enhanced by mutation
of encoded
proteins or by ubiquitination in DNA vaccination, which
will augment
the protective effects of the vaccine, as shown by
our data and others
(
33,
39,
40). Moreover, mutation of
a viral protein will
also result in abrogation of its function,
which is often harmful to
the host, further ensuring the safety
of the
vaccine.
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ACKNOWLEDGMENTS |
This work was supported by the Breast and Cervical Cancer Research
Fund (grant 83880356) from the Illinois Department of Public Health.
We thank David Stone for assistance in preparing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Microbiology and Immunology, Stritch School of Medicine,
Building 105, Room 2890, Loyola University Medical Center, 2160 South
First Ave., Maywood, IL 60153. Phone: (708) 327-3481. Fax: (708)
216-1196. E-mail: lqiao{at}luc.edu.
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Journal of Virology, September 1999, p. 7877-7881, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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