Human papillomavirus immunogen that provides protective tumor immunity and induces tumor regression.

Abstract

Human papillomavirus (HPV) is associated with premalignant lesions such as high-grade cervical intraepithelial neoplasia (CIN-III) with potential progression to cervical carcinoma. There are now preventive vaccines against HPV. However, no effective therapeutic vaccine or immunological treatment exists for individuals already infected or for the 470,000 women that develop high-grade dysplasia, carcinoma in situ, and cervical cancer each year. More than half of these women die from cervical cancer. Relative non-immunogenicity of HPV infection is one of the main reasons for the difficulty in designing a comprehensive therapeutic vaccine against HPV-induced premalignant lesions and cervical carcinoma. HPV E6 and E7 proteins, the major HPV oncogenes, are highly immunogenic but fail to induce cross-reactive and protective immune responses against heterologous strains. We designed and synthesized a therapeutic peptide vaccine comprised of multivalent peptide mixtures called hypervariable epitope constructs (HECs) that represent the major epitope variants of the oncogenic E7 structural protein, and assessed their immunogenicity and in vivo efficacy in mice. Our results show that this peptide vaccine can induce strong, HPV-specific, T-helper cell and CTL responses. More significantly, we have demonstrated that the vaccine is efficacious as a therapeutic agent in a mouse HPV tumor model. Therefore, the HPV HEC vaccine approach described herein can potentially prevent progression of HPV-associated premalignant lesions, and may also be therapeutic against tumors associated with HPV.

Full text

Page 1

Human Papillomavirus Immunogen That Provides Protective
Tumor Immunity and Induces Tumor Regression
Juan P. Marquez,1Rebecca Rivera,1Kyung Hee Kang,1Murray B. Gardner,2and Jose ´ V. Torres1
Abstract
Human papillomavirus (HPV) is associated with premalignant lesions such as high-grade cervical in-
traepithelial neoplasia (CIN-III) with potential progression to cervical carcinoma. There are now preventive
vaccines against HPV. However, no effective therapeutic vaccine or immunological treatment exists for
individuals already infected or for the 470,000 women that develop high-grade dysplasia, carcinoma in situ,
and cervical cancer each year. More than half of these women die from cervical cancer. Relative non-
immunogenicity of HPV infection is one of the main reasons for the difficulty in designing a comprehensive
therapeutic vaccine against HPV-induced premalignant lesions and cervical carcinoma. HPV E6 and E7
proteins, the major HPV oncogenes, are highly immunogenic but fail to induce cross-reactive and protective
immune responses against heterologous strains. We designed and synthesized a therapeutic peptide vaccine
comprised of multivalent peptide mixtures called hypervariable epitope constructs (HECs) that represent the
major epitope variants of the oncogenic E7 structural protein, and assessed their immunogenicity and in vivo
efficacy in mice. Our results show that this peptide vaccine can induce strong, HPV-specific, T-helper cell and
CTL responses. More significantly, we have demonstrated that the vaccine is efficacious as a therapeutic
agent in a mouse HPV tumor model. Therefore, the HPV HEC vaccine approach described herein can
potentially prevent progression of HPV-associated premalignant lesions, and may also be therapeutic against
tumors associated with HPV.
Introduction
H
cer (4,7,18). Two licensed vaccines, Gardasil and Cervarix,
have proven to be highly efficacious for the prevention of
HPV infection (3,5,27). Both vaccines are based on the in-
duction of neutralizing antibodies to recombinant major
capsid protein 1, L1, expressed as virus-like particles (5,27).
These vaccines are relatively restricted to the specific geno-
typesofHPVincludedinthevaccine(HPV16,18,6,and11)(5).
However, neither vaccine is capable of treating estab-
lished HPV infection or HPV-associated lesions (2,6,11,22,33).
Therapeutic vaccines require the induction of cell-mediated
immunity (9,33). The HPV-encoded early proteins E6 and E7
are ideal targets for therapeutic vaccines because they are
consistently expressed and are essential oncogenes in HPV-
associated precancerous lesions and cervical cancer (44,32).
Various forms of therapeutic vaccines targeting the HPV E6/
E7 antigens, often combined with other therapeutic modali-
igh-risk types of human papillomavirus (HPV) are
important agents in the pathogenesis of cervical can-
ties, have been tested in preclinical and clinical trials with
promising results (1,8,10,12,30,40,42,43). Included in these
trials, there have been peptide-based HPV vaccines consisting
of overlapping peptides covering the E6/E7 antigens, and
presenting enough epitopes to partially overcome MHC
restriction (13,16). Such peptide vaccines have been well
tolerated in preclinical and clinical trials, and have proven
capable of generating antigen-specific T-cell responses to a
broad spectrum of E6/E7 epitopes in some patients with
premalignant lesions associated with HPV (20,23,40). How-
ever, the clinical efficacy of such peptide vaccines as thera-
peutic modalities remains to be determined in patients with
carcinomainsitu,andideally thesetherapeuticvaccines could
target both premalignant lesions and cervical carcinoma
(24,28,33,35).
We report here the immunogenicity, prophylactic, and
therapeutic effects of synthetic E7 peptide constructs in an
HPV-induced murine transplantable tumor model. These
peptide constructs are unique in that they are synthesized to
represent the maximum intra-genotype variability of the E7
1Department of Medical Microbiology and Immunology, and
Laboratory Medicine, School of Medicine, University of California, Davis, California.
2Center for Comparative Medicine and Department of Pathology and
VIRAL IMMUNOLOGY
Volume 25, Number 2, 2012
ª Mary Ann Liebert, Inc.
Pp. 141–152
DOI: 10.1089/vim.2011.0066
141

Page 2

oncoprotein, and include several epitopes known to be rec-
ognized by human helper T cells. Our results show that such
hypervariable epitope constructs (HECs), given with adju-
vant, induce robust cellular and humoral immune responses
that protect mice against HPV tumor cell challenge, and that
are also able to temporarily reduce established HPV tumors.
Furthermore, we demonstrate that lymphocytes from cervi-
cal cancer patients recognize the E7 HEC peptide constructs
in vitro, suggesting that these epitopes are presented as tu-
mor antigens in vivo.
Materials and Methods
Animals and tumor cell line
Female C57BL/6 mice, 6–8wk of age, were purchased
from Charles River Laboratories. The Animal Care and Use
Committee of the University of California at Davis approved
these animal experiments. Mice were housed under specific
pathogen-free conditions. The TC-1 tumor cell line was a gift
from Dr. T.C. Wu (John Hopkins Medical Institutions, Bal-
timore, MD). TC-1 cells are derived from mouse lung epi-
thelial cells transformed to express HPV16 E6/E7 and
activated c-Ha-ras oncogene. TC-1 cells were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine
serum, 2mM L-glutamine, and 50lM 2-mercaptoethanol.
Cells were grown at 37?C and 5% CO2.
HPV hypervariable epitope construct
The sequences of the HPV HECs are shown in Table 1. The
procedure of synthesizing HECs has been described else-
where (26,31). Briefly, the possible amino acids for each po-
sition along a variable epitope are determined from
published sequence information of HPVs present globally.
Subsequently, selected amino acid coupling steps in the
synthesis of the epitope are performed with two amino acids
chosen to represent the antigenic diversity determined from
sequence data. Thus, in a single synthesis, an HEC consisting
of a mixture of peptides representing the observed in vivo
variants of the epitope is produced. HPV HECs were syn-
thesized by 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry,
cleaved from the resin, and deprotected using trifluoroacetic
acid (TFA) with EDT as scavenger, followed by extensive
dialysis and lyophilization. Amino acid analysis was per-
formed to ensure the appropriate amino acid contents, and the
purity was analyzed by reverse-phase HPLC. Peptide stocks
were prepared in 1·PBS for immunization experiments, and
for ELISA and in RPMI 1640 media for in vitro cell assays.
Immunogenicity studies
C57BL/6 mice (n=6) were vaccinated intradermally (ID)
at the base of the tail with 100lg of all three HPV HECs
(equimolar mixture) in Montanide ISA-51 (Seppic Inc., Fair-
field, NJ) four times on days 0, 12, 28, and 45. One week after
the last immunization, the mice were euthanized and cellular
immune assays were performed with cells harvested from
the spleen and lymph nodes. The number of mice per ex-
periment was chosen based on those used for several other
HPV vaccines tested in animal models (13).
Therapeutic vaccination
Mice (n=12) were injected SC in the back with 2·105TC-1
tumor cells in 200lL of 1·PBS. Twelve days later, mice with
tumors of palpable size (* 5mm) were selected and ran-
domly divided into two groups (n=6). Mice from group 1
were left unvaccinated (controls). Mice from group 2 were
vaccinated ID with 100lg of all three HPV HECs (equimolar
mixture) in Montanide ISA-51 (Seppic Inc.), followed by
boosters 12 and 28d later. Tumor development was moni-
tored at least twice a week for 130d.
Prophylactic vaccination
One week after the fourth immunization, mice (n=6) were
challenged subcutaneously (SC) in the shaved back with
1·106TC-1 tumor cells in 200lL of 1·PBS, and tumor growth
was monitored at least twice a week for a period of 150d. TC-
1 tumor cells were cultured in vitro, trypsinized, and washed
three times with 1·PBS prior to being injected into mice.
HPV HEC-specific antibody response by ELISA
One week after the third and fourth immunizations,
blood was collected and tested for HPV HEC-specific anti-
bodies by ELISA. Ninety-six-well microtiter plates were
Table 1. Sequences of Human Papillomavirus-16
(HPV-16) Hypervariable Epitope Constructs (HECs)
HEC-1. E7: 38–57
I75DG50P75NGQAEP50A75REHYN50IVTT
T25-H50H25-----Q50D25----R50----
(T helper human,
T helper mouse,
CTL mouse H-2kb)
HEC-2. E7: 10–29
DI75VLDLQ50FE75P50V50DLY75CYQLD
-Y25----E50-T25E50T50--H25-----
(T helper human)
HEC-3. E7: 73–92
H50E50DLRV50LQQ75LLMG75TLGIVCH
Q50A50---T50--D25---D25-------
(T helper human)
Each HEC is based on an epitope in the E7 protein, recognized as human T-helper cell epitope, and expected to
contain 64 variants. HPV HEC-1 also represents a murine cytotoxic T-cell epitope based on an H-2Kb
background. Letters below each sequence indicate positions where two amino acids were added, and numbers
indicate the percentage of each amino acid.
142 MARQUEZ ET AL.

Page 3

coated with 10lg/mL HPV HEC peptides per well in car-
bonate buffer (pH 9.6) overnight at 37?C. Non-specific
binding sites were blocked with PBS/0.5% v/v Tween 20
(PBST) containing 3% milk powder. Sera were diluted at
1:1000, 1:5000, and 1:10000 in PBST containing 0.5% BSA,
added to antigen-coated plates, and incubated 2h at 37?C.
After washing, HPV HEC-bound antibodies were detected
using horseradish peroxidase-conjugated secondary anti-
body (rat anti-mouse IgG; Southern Biotechnology Inc.,
Birmingham, AL) at 1:1000 dilution in PBST containing
0.5% BSA. Color development was determined following
the addition of SureBlue TMB microwell substrate (KPL).
Absorbance was measured spectrophotometrically at 605nm
with an automatic plate reader (VERSAmax; Molecular
Devices, Sunnyvale, CA) according to the manufacturer’s
instructions (SOFTmax PRO; Molecular Devices). Sera ob-
tained before immunization, termed pre-bleed, was used
as a negative control and compared with sera obtained after
immunization. Two HPV-unrelated peptides derived from
hepatitis C virus (HCV) and influenza viruses, as well as
recombinant survivin, were used as negative controls. Sera
were also tested against the buffer solution used to dissolve
the peptides to exclude the possibility that the responses
were caused by an artifact.
Ex vivo T-cell proliferation
Seven days after the final immunization, spleen and
lymph nodes (inguinal, axillary, and mesenteric) were har-
vested. Then 3·105cells from spleen and lymph nodes were
cultured in round-bottom 96-well plates with HPV HECs at a
final concentration of 1, 5, or 10lg/mL in RPMI media
supplemented with 10% FBS. HPV-unrelated peptides from
influenza or hepatitis C virus were used as negative controls
and ConA as positive control. After 48h of incubation with
peptide, 1lCi [3H] thymidine (Amersham Biosciences, Pis-
cataway, NJ) was added to each well. Following 18h of
culture, the plates were harvested using a PHD cell har-
vester, and the amount of tritiated thymidine incorporated
into proliferated cells was assessed with a Beckman LS
6000IC scintillation counter. Results were expressed as
counts per minute (CPM).
Cytotoxicity assay
Cytotoxic T cell (CTL) activity was determined using the
JAM test that measures fragmentation of DNA associated
with apoptosis (25). Each experiment was repeated three
times. One week after the last vaccination, splenocytes and
lymph nodes from vaccinated and control groups were
harvested, pooled, and cultured with an equimolar mixture
of the three HPV HEC peptides at a final concentration of
10lg/mL and 100IU/mL of recombinant murine IL-2
(R&D Systems, Minneapolis, MN) in 24-well tissue culture
plates (4·106cells per well) for 6d to generate effector cells.
[3H]-thymidine-labeled target cells were mixed with effec-
tor cells at various effector:target ratios. After 5h of incu-
bation at 37?C, cells were harvested using a PHD cell
harvester, and radioactivity associated with fragmented
DNA was assessed with a Beckman LS 6000IC scintillation
counter. Results were expressed as CPM. Spontaneous re-
lease was determined by incubating target cells in medium
alone. In this assay, reduction of counts when compared
with TC-1 indicates a positive response. The amount of
[3H]-thymidine in labeled TC-1 cells was used as an indi-
cator of total CPM that remained in the target cells before
incubation with effector cells. TC-1 tumor cells lysed by
incubation with 5% Triton X-100 were used to determine
maximum CPM.
ELISPOT for mouse cells
96-well MultiScreen HA sterile plates (Fisher Scientific,
Pittsburgh, PA) were equilibrated with 1·PBS and coated
with 10lg/mL of anti-mouse IFN-c capture antibody
(eBioscience, San Diego, CA) in 100lL of 1·PBS overnight at
4?C. Controls were processed in the absence of antibody.
After blocking for 2h with 100lL of medium (RPMI and 10%
FCS) at 37?C, freshly isolated splenocytes were added at
2.5·105cells per well in triplicate, followed by serial dilu-
tions in medium containing 10lg/mL of an equimolar
mixture of the three HPV HECs. HPV-irrelevant peptides
were used as negative controls, and positive control wells
received PMA/ionomycin (Sigma-Aldrich, St. Louis, MO), or
mouse anti-CD3 (e-Bioscience) in 200lL of complete medium.
Cultures were incubated at 37?C in 5% CO2for 24h. Cells
were removed by washing the plates six times with 1·PBS
containing 0.05% Tween 20 and once with 1·PBS alone.
Following the addition of 100lL of 1·PBS containing 5lg/
mL of biotinylated anti-mouse IFN-c antibody (eBioscience)
per well, plates were incubated 2h at 37?C. Wells were wa-
shed three times with 1·PBS and filled with 100lL of avidin-
horseradish peroxidase (1:1000 dilution; e-Bioscience) in
1·PBS. After standing 2h at room temperature, the wells
were washed three times with 1·PBS and developed for 30 to
60min with 100lL of freshly prepared 3-amino-9-ethyl car-
bazole (AEC) substrate (Sigma-Aldrich). The reaction was
stopped by rinsing the wells with ice-cold water. Spots were
quantified in an ELISPOT reader. Wells with media instead of
splenocytes were run in parallel as background controls.
Counts of negative control wells were subtracted from the
samples. A sample was scored positive if it reached a value
that was more than twice that of the control.
ELISPOT for human cells
Ninety-six well MultiScreen HA sterile plates were equil-
ibrated with 1·PBS and coated with 10lg/mL of anti-
humanIFN-ccaptureantibody(eBioscience)in100lLof1·PBS
overnight at 4?C. Controls were processed in the absence of
antibody. After blocking for 2h with 100lL of medium
(RPMI and 10% FCS) at 37?C, human PBMCs previously
thawed and conditioned were added at 500,000 cells per well
in triplicate, followed by serial dilutions in medium con-
taining 10lg/mL of an equimolar mixture of the three HPV
HECs. HPV-irrelevant peptides were used as negative con-
trols, and positive control wells received PMA/ionomycin
(Sigma-Aldrich) or human anti-CD3 (e-Bioscience) in 200lL
of complete medium. Cultures were incubated at 37?C in 5%
CO2for 48h. Cells were removed by washing the plates six
times with 1·PBS containing 0.05% Tween 20 and once with
PBS alone. Following the addition of 100lL of PBS contain-
ing 5lg/mL of biotinylated anti-human IFN-c antibody
(eBioscience) per well, plates were incubated 2h at 37?C. The
wells were washed three times with 1·PBS and filled with
100lL of avidin-horseradish peroxidase (1:1000 dilution;
PROTECTIVE TUMOR IMMUNITY AND TUMOR REGRESSION143

Page 4

e-Bioscience) in PBS. After standing 2h at room temperature,
the wells were washed three times with 1·PBS and devel-
oped for 30 to 60min with 100lL of freshly prepared AEC
substrate (Sigma-Aldrich). The reaction was stopped by
rinsing the wells with ice-cold water. Spots were quantified
inanELISPOTreader(Zeiss-VisionTechnologies,Hebron,KY).
Wells with media instead of PBMCs were run in parallel as
background controls. Counts of negative control wells were
subtracted from the samples. A sample was scored positive if
it reached a value that was more than twice that of the
control.
Clinical samples
The clinical samples studied were from HPV-infected
patients, cervical cancer patients, ovarian cancer patients,
and young healthy individuals with no detectable HPV
infection by PCR. The protocol was approved by the ethics
committee of the Health Department of The State of Sonora,
Mexico (protocol number CARES/81/09/05-CARES/SSP/
01-04-09).
HPV genotyping
HPV testing was performed in samples from vaginal
swabs by PCR using HPV-specific primers for HPV-16, -18,
-31, and -45.
Statistical analysis
The significance of differences between groups was de-
termined using a paired Student’s t-test (with a confidence
level of 95%) with GraphPad Prism software, version 5.01.
Two-tailed p values of <0.05 were considered statistically
significant.
Results
Design and construction of the three HPV HECs
Three HEC constructs representing three distinct regions
of the E7 protein of HPV-16 were synthesized (Table 1). The
sequences were selected based on previous studies showing
that they contain T-helper and CTL epitopes.
Immunization with HPV HECs induces antigen-specific
antibodies
Before performing survival trials of mice challenged with
tumor cells before and after vaccination with HPV HECs, we
studied the in vivo immunogenicity of HPV HECs in mice to
determine whether they could elicit strong, antigen-specific
humoral and cellular immune responses. HPV HEC-specific
antibodies in mice immunized with HPV HECs were de-
tected by ELISA (Fig. 1). Sera were collected after the
third immunization with all three HPV HECs and tested
individually against each HPV HEC to determine the HPV
HEC-specific antibody response. Results showed that mice
developed robust HPV HEC-specific antibody responses.
Sera from mice immunized with HPV HECs showed a strong
antibody response to the individual HPV HEC with which
they were immunized, but not to a peptide unrelated to
HPV HECs (FLU HEC). Interestingly, sera from mice im-
munized with HPV HECs also showed a strong antibody
response to recombinant E7 protein. Pre-bleed sera obtained
FIG. 1.
all three HPV HECs 7d after the third immunization; 10lg/mL of individual HPV HEC-1, -2, or -3, were used as coating
antigens to detect HPV HEC-specific antibodies. Pre-bleed sera obtained from the same mice prior to immunization, and
1lg/mL of recombinant E7 protein corresponding to HPV-16 (rE7) and 1lg/mL of recombinant survivin were used as
controls. The HPV-unrelated peptide, influenza HEC peptide (FLU-HEC), was also used as a negative control to demonstrate
that the antibody responses elicited after immunization were HPV HEC-specific. Sera were diluted to 1:1000, 1:5000, and
1:10,000 and the plates were read for optical density (OD) at 605nm. The results were expressed as mean OD values and
standard deviation of triplicate wells.
Induction of HPV HEC-specific antibodies. Sera were collected from mice immunized with an equimolar mixture of
144MARQUEZ ET AL.

Page 5

prior to immunization did not show antibody response to
HPV HECs.
HPV HECs induce antigen-specific
T-cell proliferative responses
We also assessed whether HPV HECs could induce
antigen-specific lymphoproliferation (Fig. 2A and B).
The results show that all six individual mice immunized
with HPV HECs developed strong HPV HEC-specific
proliferativeT-cellresponses.
spleens and lymph nodes in mice immunized with HPV
HECs proliferated, reacting to individual HPV HEC, but
there were no proliferative responses detected with
HPV-unrelated peptides, such as FLU-HEC and HCV-
HEC. Interestingly, the proliferative response to HPV
Lymphocytes from
FIG. 2.
from mice immunized with an equimolar mixture of all three HPV HECs were harvested. HPV-unrelated HECs based on a
variable epitope present in the hemagglutinin of influenza virus (FLU-HEC) and hepatitis C virus (FLU-HEC) were used as
negative controls, and ConA was used as positive control. 3·105cells were re-stimulated with individual HPV HEC or HPV-
unrelated peptides in a final concentration of 5lg/mL (white bars) or 10lg/mL (black bars). After 48h of incubation, 1lCi of
[3H]-thymidine was added, and the cells were incubated further for 18h. The amount of incorporated [3H]-thymidine into
proliferating cells was expressed as counts per minute (CPM). Data shown in this bar graph represent the results from six
individual mice with standard deviation (SD). Results with immunized (A) and non-immunized (B) animals are presented.
HPV HEC-specific proliferative T-cell responses. One week after the last immunization, spleens and lymph nodes
PROTECTIVE TUMOR IMMUNITY AND TUMOR REGRESSION145

Page 6

HEC-2 was stronger than that to HPV HEC-1 or -3, a
result similar to that observed in the detection of HPV
HEC-specific antibody response in Fig. 1. Overall, these
results suggest that the proliferative responses were
HPV HEC-specific.
HPV HECs induce specific T cells that secrete IFN-c
We also assessed the production of interferon-c (IFN-c) by
HPV HEC-specific memory T cells from mice immunized
with HPV HECs by ELISPOT analysis (Fig. 3A and B).
FIG. 3.
group) immunized four times with HPV HEC-1, -2, and -3 (an equimolar mixture of the 3 HECs). One week after the final
immunization, 10lg/mL of HPV HEC-1, -2, and -3, and irradiated TC-1 cells were added in triplicate to each well pre-coated
with anti-mouse IFN-c, and incubated for 24h. Cells containing medium only were used as background, HCV HEC as negative
control, and PMA/ionomycin as positive control. Results with immunized (A) and non-immunized (B) animals are shown.
ELISPOT detection of IFN-c production by HPV-specific T cells. Spleen cells were harvested from C57BL/6 mice (8 per
146 MARQUEZ ET AL.

Page 7

Splenocytes from mice immunized with HPV HECs were
stimulated individually with HPV HEC-1, -2, or -3. In
addition, splenocytes were also incubated with irradiated
TC-1 tumor cells to assess whether HPV HEC-primed T cells
could recognize HPV-16 E7 oncoprotein on TC-1 tumor
cells, and secrete tumor-specific IFN-c. Medium alone or
HPV-unrelated HCV HEC peptides were used as negative
controls.
Our results showedthat HPVHECs elicitedIFN-c-secreting,
HPV HEC-specificT cellsinmiceimmunizedwithHPVHECs.
Moreover, splenocytes from mice immunized with HPV HECs
also secreted IFN-c upon culture with TC-1 tumor cells. The
number of IFN-c spots produced by splenocytes stimulated
with TC-1 tumor cells was comparable to that from cells
stimulatedwithHPV HEC-1 or-3. Furthermore, thenumberof
cells secreting IFN-c in response to HPV HEC-2 was higher
than that to other HPV HECs or TC-1 cells, and was compa-
rable to the PMA/ionomycin used as positive control.
HPV HECs induce CTLs that kill tumor cells
Next we assessed whether HPV HECs could induce CTLs
that were cytotoxic to tumor cells. When cells from mice
immunized with an equimolar mixture of HPV HECs were
tested against TC-1 tumor cells, generation of HPV HEC-
specific CTLs was demonstrated, as shown in Fig. 4. CTLs
from mice immunized with HPV HECs recognized TC-1 cells
as targets, and had significant cytotoxicity against these tu-
mor cells. When the E:T ratio was 50:1, the cytotoxic re-
sponses of HPV HEC-specific CTLs were as strong as that of
the positive control. Moreover, about 25% of killing activity
of CTLs induced by HPV HECs was observed at the lowest
E:T ratio of 6:1. These data suggest that HPV HECs induce
CTLs in mice that are HPV-specific and have strong cytolytic
activity against TC-1 tumor target cells. The experiment was
performed three times.
Tumor-bearing mice can recognize
HPV HECs as antigens
Our in vivo immunogenicity study showed that HPV
HECs are strong immunogens that can elicit HPV HEC-
specific humoral and cellular immune responses. Next we
wanted to determine whether natural tumor-specific im-
mune responses generated in tumor-bearing mice could
recognize HPV HECs. C57BL/6 mice (n=6) were injected
with 1·106TC-1 tumor cells. After 21d, all the mice devel-
oped a tumor larger than 1.5cm in diameter. The spleens
were harvested, and total splenocytes (3·105cells per well)
were cultured in vitro with 10lg/mL HPV HEC or a mix-
ture of HPV HEC-1, -2, and -3. Media alone or HPV HEC-
unrelated peptides (HCV-HEC) were used as negative
controls.
The proliferative responses of lymphocytes from HPV-16
E7-positive tumor-bearing mice were specific for HPV HECs,
showing negative responses to HPV HEC-unrelated peptide
(HCV-HEC), and to media only. This was an interesting re-
sult, in that immune responses generated against TC-1
FIG. 4.
were harvested and a CTL assay was performed in triplicate using the JAM test. Results were derived from pooled lymph
node and spleen cells cultured at a final concentration of 10lg/mL of a mixture of three HPV HECs and IL-2 (500IU/mL) for
6d to generate effector killer cells. HPV HEC-specific effector cells were added to [3H]-thymidine-labeled (5lCi/mL) TC-1
tumor cells at different ratios of effectors to targets: 50:1, 25:1, 12:1, and 6:1. The amount of [3H]-thymidine from live target
cells that were not killed by CTLs was measured by the JAM test to determine the cytotoxicity of HPV HEC-specific CTLs.
The amount of [3H]-thymidine in labeled TC-1 cells alone was used as an indicator of the total CPM that remained in target
cells before incubation with effector cells. TC-1 tumor cells incubated with 5% Triton X-100 were used as positive control for
maximum lysis.
CTL responses in HPV HEC-immunized mice. One week after the last immunization, splenocytes and lymph nodes
PROTECTIVE TUMOR IMMUNITY AND TUMOR REGRESSION 147

Page 8

tumors had reactivity against HPV HECs in vitro. These re-
sults suggest that even under immunosuppressive condi-
tions, there were spontaneous T cells able to react with the
synthetic HPV constructs. This may represent spontaneous
but irrelevant immune responses due to the limitations of the
model, and/or that the large number of tumor cells trans-
planted did not stop tumor growth. It is possible that these
spontaneous T cells potentially represent the capability of the
peptides to be presented in vivo. The data suggest that the
HPV HECs represented the epitopes in the tumor. In addi-
tion, the lymphoproliferative response to HPV HEC-2 from
TC-1 tumor-bearing mice was the strongest of the three HPV
HECs tested (Fig. 5). These results are consistent with the
experiments showing that the in vivo immunogenicity of
HPV HEC-2 was the strongest in terms of antibody response,
lymphoproliferative response, and IFN-c detection by HPV
HEC-specific T cells. These results suggest that HPV HEC-2
could be the strongest immunogenic CD4+T-cell epitope.
Lymphocytes from patients with cervical carcinoma
in situ and CIN 2/3 recognize the HPV HECs
We also examined a repository of PBMC samples from
HPV-positive patients with pre-malignant lesions (CIN 2/3)
and cervical carcinoma to determine whether HPV HECs
could be recognized by cells from this group of patients
(Fig. 6). The samples were collected just before initiation of
standard of care treatment. Repository samples from HPV-
negative patients, and from patients with ovarian carcinoma
(HPV-negative), were used as controls. HPV HEC-2, the
most immunogenic peptide based on our in vivo immuno-
genicity study of HPV HECs in mice, was tested in vitro to
assess whether HPV HEC-2 stimulation could elicit IFN-c
secretion by lymphocytes from these patients. Our results
show that lymphocytes from patients with pre-malignant
lesions (CIN 2/3) and cervical carcinoma secreted IFN-c
upon stimulation with HPV HEC-2, suggesting that HPV-
positive patients from both groups can recognize HPV HEC-2
as antigen presented in vivo.
HPV HECs can be effective as a therapeutic vaccine
To determine the in vivo efficacy of HPV HECs as a ther-
apeutic vaccine, the transplantable TC-1 tumor cell line ex-
pressing HPV E7 was used to challenge mice before
vaccination (Fig. 7). Mice started developing tumors that
were palpable 12d after injection. Mice were randomly di-
vided into two groups (6 mice per group). In the group of
mice that was vaccinated after tumor challenge, tumor
growth was stabilized and slowed down, never growing
more than 0.5cm in size. Furthermore, starting around day
60 post-inoculation, tumor regression was observed, and
mice that received the therapeutic HPV HEC vaccine became
tumor-free at about 105d after challenge. In contrast to mice
that received therapeutic HPV HEC vaccination, unvacci-
nated mice had to be sacrificed 20d after challenge with
tumor cells due to large tumor growth.
HPV HECs can be effective as a prophylactic vaccine
After demonstrating the therapeutic activity of the HPV
HEC construct, we determined the efficacy of HPV HEC as a
prophylactic vaccine (Fig. 8). Seven days after the last im-
munization, mice were challenged subcutaneously with
1·106TC-1 tumor cells. Tumor growth and mouse survival
were monitored for 150d. Unvaccinated mice of the same
age as the vaccinated group were used as controls. All mice
in the control group developed progressive tumors and were
eventually sacrificed before 30d due to tumor growth
FIG. 5.
tumor cells. Spleens were harvested from mice that developed a tumor larger than 1.5cm in diameter after 21d. 3·105
splenocytes were incubated in vitro with individual HPV HECs or an equimolar mixture of all three HPV HECs at a final
concentration of 10lg/mL. Proliferative responses specific to HPV HECs were assessed by measuring the amount of [3H]-
thymidine incorporated by cells during proliferation. The results were expressed as mean counts per minutes (CPM) with
standard deviation in triplicate.
Splenocytes from tumor-bearing mice recognize the HPV HECs in vitro. Mice (n=6) were injected with 1·106TC-1
148 MARQUEZ ET AL.

Page 9

beyond the size restriction of the approved protocol. Re-
markably, all the animals vaccinated with HPV HECs were
protected from tumors up to 65d, remaining tumor-free.
Although some mice eventually developed delayed tumor
formation starting at 70d after challenge, this was only seen
in 25% of the vaccinated animals. Moreover, more than 60%
of mice remained tumor-free until the end of the prophylactic
study (150d).
Discussion
In this report, we have shown that an immunogen based
on three antigenically variable epitopes of the E7 protein of
HPV can be used both therapeutically and preventively
against challenge with tumor cells expressing E7. These re-
sults demonstrate that HPV HECs are strong immunogens
inducing antibodies and T cells that are specific to tumors
expressing HPV E7 protein, and importantly that these
peptides are recognized by PBMCs from cervical carcinoma
patients.
Multiple HPV immunotherapy clinical trials have in-
volved immunization with virus-like particles (VLPs), DNA,
peptides, and adoptive therapy with dendritic cells (DCs)
pulsed with peptides (14,15,19,21,37,38,40). Most have been
Phase I trials showing safety of the vaccine formulation
(12,37,38,40,41). Knowledge gained from clinical trials per-
formed to date indicates that neutralizing antibodies and/or
E6/E7-specific CTLs can be produced in healthy subjects,
and to an appreciable extent in patients with cervical dys-
plasia (9,16,34,39).
The role of cellular immune responses mediated by CTLs
in protection against progression of CIN2/3 to carcinoma
in situ has been extensively examined (16,17,20). It is clear
that HPV-16-specific CTLs can be detected in the blood of
these patients (32,34,40,41).
Relatively low immunogenicity and intra-strain vari-
ability complicate the development of a broadly protective
and effective therapeutic vaccine against pre-malignant le-
sions and cervical carcinoma associated with the main types
and subtypes of HPV (35,37). A hallmark of the HPV virus
is its extensive intra-strain antigenic variation (2,3,6). Al-
though HPV antigenic epitopes show sequence variation,
we have observed that the sequence diversity of these epi-
topes appears to be limited and partially predictable
(16,18,21). To exploit this pattern of diversity, we have de-
veloped a procedure that uses computer-aided epitope
variation analysis and peptide synthesis to produce a pep-
tide mixture representing the known amino acid sequences
of an antigenic epitope (26,31). This approach of re-
presenting the known antigenic variant sequences of an
epitope was developed at our laboratory, and the synthetic
immunogen was named hypervariable epitope construct
(HEC) (26). These immunogens are then validated for an-
tigenicity with a collection of sera obtained from affected
individuals, such as those bearing CIN 2/3, carcinoma
in situ, and cervical carcinoma (31).
The experiments reported here included an immunoge-
nicity study of HPV HECs, anti-tumor T-cell recognition of
tumor cells expressing E7, in vivo efficacy of HPV HECs for
prevention of tumor establishment, and as therapy of
FIG. 6.
initiation of standard of care treatment from HPV-16/HPV-18-positive patients with CIN 2/3 [HPV(+)/CIN], or cervical
carcinoma [HPV(+)/CC] recognize the most immunogenic HPV HEC peptide, HEC-2. Cells from 50 patients were tested by
ELISPOT. The ovarian cancer (Ovarian CA) patients selected were negative for HPV.
IFN-c secretion by lymphocytes from HPV-infected patients incubated with HPV HEC-2. Cells collected just before
PROTECTIVE TUMOR IMMUNITY AND TUMOR REGRESSION149

Page 10

existing tumors and the assessment of the spontaneous im-
mune response in cervical cancer patients.
The immunogenicity studies demonstrated that HPV
HECs are capable of inducing HPV HEC-specific humoral
and cellular immune responses. Antibodies generated in
mice strongly bound only to HPV HECs and recombinant E7
and did not show cross-reactivity with other peptides and
proteins unrelated to HPV (Fig. 1). The data suggest that
HPV HECs induce B cells that produce abundant antibodies
capable of recognizing HPV HECs and the recombinant
FIG. 8.
equimolar mixture of all three HPV HECs four times at days 0, 12, 28, and 45. Seven days after the last immunization, the
mice were challenged SC with 1·106TC-1 tumor cells. Tumor growth and survival of mice were monitored for 150d.
Unvaccinated mice of the same age (squares) were also challenged with the same number of tumor cells and used as a control
group. A significant number of the mice remained tumor free for more than 130d. In 25% of the animals, tumors re-appeared
after 70 days (triangles). Data are presented as the mean of 6 mice per group.
Preventive immunization induces tumor stabilization and regression. Mice were vaccinated ID with 100lg of an
FIG. 7.
TC-1 tumor cells. Palpable tumors were detected 12d later, and mice were divided into two groups. Mice in the experimental
group (triangles) were vaccinated with 100lg of an equimolar mixture of all three HPV HECs, followed by boosters with the
same amount 12 and 28d later. Control group mice (squares) were left unvaccinated. Tumor growth of mice from tumor
challenge were monitored for up to 130d. Data are presented as the mean of 6 mice per group.
Therapeutic effect of the HPV HEC vaccine. Eight- to 10-wk-old female mice (n=12) were injected SC with 2·105
150 MARQUEZ ET AL.

Page 11

HPV-16 E7 protein with high affinity. In a future study, it
would be interesting to learn how long HPV HEC-specific
antibodies would persist after the final immunization.
The cellular immune responses induced by HPV HEC in
mice were quite interesting. Proliferation responses and IFN-c
secretion of T cells were significantly strong and specific to
HPV HECs and recombinant E7 protein (Fig. 2). The results
presented here suggest that robust proliferative T-cell re-
sponses can also be induced in mice immunized with HPV
HECs. As this lymphoproliferation assay was performed
with unfractionated lymphocytes, it is also possible that
antigen-presenting DCs that process HPV HECs could cross-
present to CD8+T cells and induce HPV HEC-specific CD8+
T-cell proliferative responses. Further studies are required to
explore this possibility.
It was surprising to detect IFN-c secretion when T cells
from HPV HEC-immunized mice were incubated with tu-
mor cells (Fig. 3A). In addition, mice injected with tumor
cells developed strong spontaneous proliferative splenocyte
responses to HPV HECs. These results suggest that although
the natural immune response only represents the pool of T
cells generated against the tumor cells, there were also
memory cells able to recognize the HEC peptides (Fig. 5). In
fact, there are several reports concerning the study of the
natural/spontaneous immune response in cancer-bearing
hosts, even under the high tumor burdens seen when the
pool of cells that were generated against the tumor eventu-
ally became irrelevant due the multiple immunosuppressive
networks (29,36).
These responses were specific for HPV HECs, and no
significant responses to HPV-irrelevant peptides or proteins
were observed. These results could indicate that the peptides
from HPV HECs were intracellularly processed, and that
they include sequences that are very similar to the naturally-
processed epitopes derived from tumor cells. These data
suggest that HPV HECs represent important antigenic epi-
topes of E7 found in HPV tumors, and are strong immuno-
gens that induce antigen-specific cellular responses.
It was significant to determine that the ex vivo-generated
effector killer cells from mice immunized with HPV HECs had
strong cytotoxic activity against TC-1 tumor target cells (Fig.
4). Results from the JAM test suggest that HPV HECs can
induce robust CTLs against TC-1 tumor cells in vivo in mice.
Based on these positive results, we wanted to determine if
tumor-specific naturally-occurring immune responses gen-
erated in humans with CIN 2/3 and cervical carcinoma and
in HPV tumor-bearing mice could recognize the most im-
munogenic HPV HEC (HEC-2).
Significantly, lymphocytes from cervical cancer patients
and CIN 2/3 patients secrete IFN-c when incubated with
these immunogens (Fig. 6). These data may suggest that
some of these peptides are naturally processed in vivo, and
may represent good candidates for future human studies
using HEC peptides as potential therapeutic vaccines.
The proliferative response to HPV HEC-2 in tumor-bearing
mice was the strongest among the three HPV HECs tested,
and this correlates with our findings with the clinical samples.
This is consistent with the results obtained when studying the
immunogenicity of HPV HECs, showing that proliferative
responses to HPV HEC-2 were stronger than to the other two
HECs. It is reasonable to conclude that HPV HEC-2 represents
the strongest immunogenic peptide. These results suggest that
the sequences represented by HPV HECs are processed in vivo
and presented to the immune system as natural epitopes.
Mice were protected from tumors when they were vacci-
nated with HPV HECs before (Fig. 7) and after tumor chal-
lenge (Fig. 8). Not only were mice able to survive tumor
challenge, but the size of established tumors was also reduced.
Collectively, these data suggest that HPV HECs, a mixture
of peptides designed by our laboratory based on the anti-
genic variation of the E7 oncoprotein, turned out to be both
an effective preventive and therapeutic vaccine, with the
potential to act against pre-malignant lesions and tumors.
The vaccine is inexpensive to produce and very stable, and
can be stored in dry powder form (lyophilized) at room
temperature for use in developing countries without the
need for cold chain storage and distribution.
Acknowledgments
We thank Carmelo R. Peraza and Dylan Dodd for expert
technical work.
Author Disclosure Statement
No competing financial interests exist.
References
1. Katsuyuki A, Kawana K, Yokoyama T, et al.: Oral immuni-
zation with a Lactobacillus casei vaccine expressing human
papillomavirus (HPV) type 16 E7 is an effective strategy to
induce mucosal cytotoxic lymphocytes against HPV16 E7.
Vaccine 2010;28:2810–2817.
2. Albers A, and Kaufmann AM: Therapeutic human papillo-
mavirus vaccination. Public Health Genomics 2009;12:331–342.
3. Arie S: Global HPV vaccination. BMJ 2011;342:d1042.
4. Astbury K, and Turner MJ: Human papillomavirus vacci-
nation in the prevention of cervical neoplasia. Int J Gynecol
Cancer 2009;19:1610–1613.
5. Bach P: Gardasil: from bench, to bedside, to blunder. Lancet
2010;375:963–964.
6. Bermudez-Humaran L, and Langella P: Perspectives for the
development of human papillomavirus vaccines and im-
munotherapy. Expert Rev Vaccines 2010;9:35–44.
7. Bertuccio MP, Spataro P, Caruso C, and Picerno I: Detection
of human papillomavirus E6/E7 mRNA in women with
high-risk HPV types 16, 18, 31, 33 and 45 which are asso-
ciated with the development of human cervical cancer. Eur J
Gynaecol Oncol 2011;32:62–64.
8. Best S, Peng S, Juang CM, et al.: Administration of HPV
DNA vaccine via electroporation elicits the strongest CD8+
T cell immune responses compared to intramuscular injec-
tion and intradermal gene gun delivery. Vaccine 2009;27:
5450–5459.
9. Bhat P, Mattarollo SR, Gosmann C, Frazer IH, and Leggatt
GR: Regulation of immune responses to HPV infection and
during HPV-directedimmunotherapy.
2011;239:85–98.
10. Bijker M, van den Eeden SJ, Franken KL, Melief CJ, Offringa
R, and van der Burg SH: CD8+ CTL priming by exact
peptide epitopes in incomplete Freund’s adjuvant induces a
vanishing CTL response, whereas long peptides induce
sustained CTL Reactivity. J Immunol 2007;179:5033–5040.
11. Bolhassani A, Mohit E, and Rafati S: Different spectra of
therapeutic vaccine development against HPV infections.
Hum Vaccine 2009;5:671–689.
Immunol Rev
PROTECTIVE TUMOR IMMUNITY AND TUMOR REGRESSION 151

Page 12

12. Brun J-L, Dalstein V, Leveque J, et al.: Regression of high-
grade cervical intraepithelial neoplasia with TG4001 tar-
geted immunotherapy. Am J Obstet Gynecol 2011;204:
169e1–169e8.
13. Chao-Yi W, Archana M, Xiaowu P, Chien-Fu H, and T-C
Wu: Improving therapeutic HPV peptide-based vaccine
potency by enhancing CD4+ T help and dendritic cell acti-
vation. J Biomed Sci 2010;17:88.
14. Chuang C-M, Monie A, Wu A, and Hung CF: Combination
of apigenin treatment with therapeutic HPV DNA vaccina-
tion generates enhanced therapeutic antitumor effects.
J Biomed Sci 2009;16:49.
15. Decrausaz L, Revaz V, Bobst M, Corthesy B, Romero P, and
Nardelli-Haefliger D: Induction of human papillomavirus
oncogene-specific CD8 T-cell effector responses in the genital
mucosa of vaccinated mice. Int J Cancer 2010;126:2469–2478.
16. De Lorenzo BHP, Ramos MC, Michelin MA, and Murta EF:
Progress in the use of immunotherapy to treat uterine cer-
vical cancer. Tumori 2009;95:1–7.
17. Frazer I: Measuring serum antibody to human papilloma-
virus following infection or vaccination. Gynecol Oncol
2010;118:S8–S11.
18. Hellner K, and Munger K: Human papillomaviruses as
therapeutictargetsinhuman
2011;29:1785–1794.
19. Hoffmann C, Stanke J, Kaufmann AM, Loddenkemper C,
Schneider A, and Cichon G: Combining T-cell vaccination
and application of agonistic anti-GITR mAb (DTA-1) in-
duces complete eradication of HPV oncogene expressing
tumors in mice. J Immunother 2010;33:136–145.
20. Hung C-F, Wu TC, Monie A, and Roden R: Antigen-specific
immunotherapy of cervical and ovarian carcinoma. Im-
munological Rev 2008;222:43–69.
21. Kanduc D: Quantifying the possible cross-reactivity risk of
an HPV16 vaccine. J Exp Ther Oncol 2009;8:65–76.
22. Kietpeerakool C and Srisomboon J: Medical treatment of
cervical intraepithelial neoplasia II, III: an update review. Int
J Clin Oncol 2009;14:37–42.
23. Lin K, Doolan K, Hung CF, and Wu TC: Perspectives for
preventive and therapeutic HPV vaccines. J Formos Med
Assoc 2010;109:4–24.
24. Lin K, Roosinovich E, Ma B, Hung CF, and Wu TC: Ther-
apeutic HPV DNA vaccines. Immunol Res 2010;47:86–112.
25. Matzinger P: The JAM test. A simple assay for DNA
fragmentation and cell death. J Immunol Methods 1991;145:
185–192.
26. Meyer D and Torres JV: Hypervariable epitope construct: a
synthetic immunogen that overcomes MHC restriction of
antigen presentation. Molec Immunol 1999;36:631–637.
27. Nicol A, Nuovo GJ, and Dillner J: A summary of the 25th
International Papillomavirus Conference 2009: vaccines,
screening, epidemiology and therapeutics. J Clin Virol
2010;47:208–215.
28. Nieto K, Gissmann L, and Schadlich L: Human papilloma-
viruses-specific immune therapy: failure and hope. Antivir
Ther 2010;15:951–957.
29. Ohue Y, Eikawa S, Okazaki N, et al.: Spontaneous antibody
and CD4 and CD8 T-cell responses against XAGE-1b (GA-
GED2a) in non-small cell lung cancer patients. Int J Cancer
2011.
30. Peng S, Monie A, Pang X, Hung CF, and Wu TC: Vascular
disrupting agent DMXAA enhances the antitumor effects
generated by therapeutic HPV DNA vaccines. J Biomed Sci
2011;8:18–21.
cancer.JClinOncol
31. Reddy J, Banapour B, Anderson DE, Lee SH, Marquez JP,
Carlos MP, and Torres J: Induction of immune responses
against human papillomavirus by hypervariable epitope
constructs. Immunology 2004;112:321–327.
32. Santin A, Bellone S, Palmieri M, et al.: HPV16/18 E7-pulsed
dendritic cell vaccination in cervical cancer patients with
recurrent disease refractory to standard treatment modali-
ties. Gynecologic Oncol 2006;100:469–478.
33. Su J-H, Wu A, Scotney E, Ma B, Monie A, Hung CF, and Wu
TC: Immunotherapy for cervical cancer: Research status and
clinical potential. BioDrugs 2010;24:109–129.
34. Trimble C, Peng S, Thoburn C, Kos F, and Wu TC: Naturally
occurring systemic immune responses to HPV antigens do
not predict regression of CIN2/3. Cancer Immunol Im-
munother 2009;59:799–803.
35. Trimble C and Frazer IH: Development of therapeutic HPV
vaccines. Lancet Oncol 2009;10:975–980.
36. Tsuji T, Matzuzaki J, Ritter E, et al: Split T cell tolerance
against a self/tumor antigen: spontaneous CD4+ but not
CD8+ T cell responses against p53 in cancer patients and
healthy donors. PLoS One 2011;6:e23651.
37. Tuma R: Cervical cancer: the second-generation vaccines
move forward. J Natl Cancer Inst 2009;101:774–775.
38. Van der Burg S, Arens R, and Melief CJ: Immunotherapy for
persistent viral infections and associated disease. Trends
Immunol 2011;32:97–103.
39. Van der Burg S and Palefsky JM: Human immunodeficiency
virus and human papilloma virus—why HPV-induced le-
sions do not spontaneously resolve and why therapeutic
vaccination can be successful. J Transl Med 2009;7:108.
40. Van Doorslaer K, Reimers LL, Studentsov YY, Einstein MH,
and Burk RD: Serological response to an HPV16 E7 based
therapeutic vaccine in women with high-grade cervical
dysplasia. Gynecol Oncol 2010;116:208–212.
41. Welters M, Kenter GG, Piersma SJ, et al.: Induction of tumor-
specific CD4+ and CD8+ T-cell immunity in cervical cancer
patients by human papillomavirus type 16 E6 and E7 long
peptides vaccine. Clin Cancer Res 2008;14:178–187.
42. Welters M, et al.: Success or failure of vaccination for HPV16-
positive vulvar lesions correlates with kinetics and pheno-
type of induced T-cell responses. Proc Natl Acad Sci USA
2010;107:11895–11899.
43. Yan J, Harris K, Khan AS, Draghia-Akli R, Sewell D, and
Weiner DB: Cellular immunity induced by a novel HPV18
DNA vaccine encoding an E6/E7 fusion consensus protein
in mice and rhesus macaques. Vaccine 2008;26:5210–5215.
44. Zwaveling S, et al.: Established human papillomavirus
type 16-expressing tumors are effectively eradicated fol-
lowing vaccination with long peptides. J Immunol 2002;169:
350–358.
Address correspondence to:
Dr. Jose ´ V. Torres
Department of Medical Microbiology and Immunology
School of Medicine
Tupper Hall, Room 3226
University of California
Davis, CA 95616
E-mail: jvtorres@ucdavis.edu
Received September 19, 2011; accepted December 20, 2011.
152MARQUEZ ET AL.