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Vol. 9, 5205–5213, November 1, 2003
Clinical Cancer Research 5205
Vaccinia-Expressed Human Papillomavirus 16 and 18 E6 and E7
as a Therapeutic Vaccination for Vulval and Vaginal
Intraepithelial Neoplasia
Peter J. Baldwin, Sjoerd H. van der Burg,
Christopher M. Boswell, Rienk Offringa,
Julian K. Hickling, Jennifer Dobson,
John St. Clair Roberts, John A. Latimer,
Robin P. Moseley, Nicholas Coleman,
Margaret A. Stanley, and Jane C. Sterling1
Departments of Gynaecological Oncology [P. J. B., J. A. L.],
Dermatology [J. C. S.], and Pathology [R. P. M.], Addenbrooke’s
National Health Service Trust, Cambridge CB2 2QQ, United
Kingdom; Department of Pathology, University of Cambridge,
Cambridge CB2 1QP, United Kingdom [P. J. B., N. C., M. A. S.,
J. C. S.]; Xenova Group plc., Cambridge CB4 0WG, United Kingdom
[C. M. B., J. K. H., J. D., J. S. C. R.]; and Immunohematology and
Blood Transfusion, Leiden University Medical Center, 2333 ZA
Leiden, the Netherlands [S. H. v. d. B., R. O.]
ABSTRACT
Purpose: Anogenital intraepithelial neoplasia is a
chronic disorder associated with infection by high-risk human papillomavirus (HPV) types. It is frequently multifocal
and recurrence after conventional treatment is high. Boosting HPV-specific cell-mediated immune responses may reduce progression to carcinoma and could lead to disease
clearance. We have tested the safety, immunogenicity, and
efficacy of a recombinant vaccinia candidate vaccine (TAHPV) in women with anogenital intraepithelial neoplasia.
Experimental Design: Twelve women, aged 42–54 years
with high-grade HPV-positive vulval or vaginal intraepithelial neoplasia of up to 15 years duration, completed a Phase
II study of TA-HPV, a live recombinant vaccinia virus,
expressing modified versions of the E6 and E7 open reading
frames from HPV-16 and HPV-18.
Results: The vaccine was well tolerated. Five of 12
(42%) patients showed at least a 50% reduction in total
lesion diameter over 24 weeks with 1 patient showing complete regression of her lesion. Overall, 83% of women
showed some improvement with an average decrease in
Received 11/21/02; revised 5/12/03; accepted 5/14/03.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
This work was supported by grants from Trinity College, Cambridge (to
P. B.), Xenova Research Ltd. (to P. B.), the Dutch Cancer Society Grant
RUL 99-2024 (to S. H. v. d. B.), and the Cancer Research Institute (to
S. H. v. d. B).
1
To whom requests for reprints should be addressed, at Division of
Virology, Department of Pathology, University of Cambridge, Tennis
Court Road, Cambridge CB2 1QP, United Kingdom. Phone: 44-1223336915; Fax: 44-1223-336926; E-mail: [email protected].
lesion size of 40%. All cases showed an increased IgG titer
and T-cell response to the vaccinia virus. An IFN-␥ enzymelinked immunospot assay using pooled 22-mer peptides
spanning HPV-16 E6 and E7 showed an increased specific
T-cell response after vaccination in 6 of the 10 cases available for testing. There was no increase in specific cytotoxic
response to selected individual HLA class I-restricted
HPV-16 E6/7 peptides.
Conclusions: The results suggest that the vaccine may
have an effect on HPV-positive vulval intraepithelial neoplasia/vaginal intraepithelial neoplasia and that additional
studies are warranted to develop an effective therapeutic
vaccine.
INTRODUCTION
Squamous intraepithelial neoplasia is common to several
anogenital sites and carries a risk of progression to invasive
SCC.2 Both intraepithelial neoplasia and SCC are commonly
associated with HR-HPVs. Indeed, for cervical disease, the
development of sensitive PCR methodology has demonstrated
HR-HPV to be present in almost all cases of SCC (1), and
HR-HPV infection is now widely accepted as a necessary cause
for cervical cancer (2). Over 85 HPV types have been fully
sequenced, and many other potential types have been identified
in mucosal and cutaneous tissue (3). Interestingly, the vast
majority of cases of cervical neoplasia are attributable to a small
subset of HR-HPV types (namely types 16, 18, 31, 33, 45, and
56; Refs. 1, 4). Persistent HR-HPV infection appears to be
required for the development of CIN (5) and may increase the
risk of disease progression at this site (6). HPV DNA is found
in 80 –100% of biopsies of high-grade VIN (grade II–III; Refs.
7, 8) and VAIN (8, 9). Classically, both HPV-dependent and
-independent SCC of the vulva have been previously described
(10), with younger women showing a high prevalence (85%) of
HR-HPV infection (11). In common with cervical neoplasia, a
limited group of HR-HPVs are associated with vulvar neoplasia,
with ⬎90% of cases involving HPV types 16, 18, or 33 (8, 12).
AGIN at sites other than the cervix poses a challenging
therapeutic problem for two reasons. Firstly, the disease is
increasing in incidence, especially in the vulval area of premenopausal women (13, 14) in whom lesions are frequently HPV
positive. Secondly, the disease is difficult to manage. Many
2
The abbreviations used are: SCC, squamous cell carcinoma; HPV,
human papillomavirus; HR-HPV, high-risk HPV; CIN, cervical intraepithelial neoplasia; VIN, vulval intraepithelial neoplasia; VAIN, vaginal intraepithelial neoplasia; AGIN, anogenital intraepithelial neoplasia; AIN, anal intraepithelial neoplasia; high-grade, histological grade II
and III; ELISPOT, enzyme-linked immunospot; OD, optical density;
PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin.
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5206 Therapeutic HPV-16 E6/E7 Vaccination for VIN/VAIN
treatments are in current use for AGIN. Surgical excision or
ablation of affected skin is the most successful way of treating
the disease, but because AGIN is commonly multifocal and
sometimes multicentric, this approach may be extensive and
disfiguring and can carry considerable psychosexual morbidity.
Recurrence rates are high, with a reported return of high-grade
VIN in ⬃50% of patients (15, 16), usually within the first 4
years after treatment. Furthermore, surgical excision does not
eliminate the risk of progression to invasive SCC, which remains of the order of 5% even after treatment (15–17). If
untreated, a small study of only eight cases has suggested a
much higher progression rate of 88% (17).
The high prevalence of HR-HPV infection in AGIN has
lead to the suggestion that therapy for the disease should aim at
the immunological eradication of virus-infected cells. Local
immunotherapy has already been described with some encouraging results. IFN has been reported to induce short-term disease regression (18) and local immune modulation with imiquimod has also led to disease clearance (19). An alternative
approach is to induce a systemic cell-mediated immune response with the aim of effecting local cytotoxicity against the
virus-infected cells. This has been attempted in cervical HPVassociated malignant and premalignant disease (20 –23) with
successful induction of a HPV-specific immune response but
little, if any, effect on the disease.
In this Phase II study, the safety and efficacy of a candidate
therapeutic vaccine against HPV (TA-HPV; Xenova Research
Ltd.) was tested in women with VIN or VAIN. All women had
high-grade disease, and the clinical effect was assessed by
measurement of the visible areas of abnormal skin or mucosa.
PATIENTS AND METHODS
Subjects
Women between the ages of 18 and 60 with high-grade
VIN, VAIN, or AIN were assessed for inclusion in the trial.
They were diagnosed by histological interpretation of a biopsy
and screened for hematological, biochemical, and immunological abnormalities by blood testing. Women were not considered
for inclusion if the use of live vaccinia virus could be expected
to be detrimental either to their health or to the health of their
close contacts. Thus, women with any degree of immunosuppression (including those receiving therapeutic immunosuppressive agents), a history of severe allergic reaction, active atopic
eczema, or other widespread inflammatory or exfoliative skin
disease were not considered for the trial. In addition, women
were excluded from consideration for vaccination if they were
in close contact with children under the age of 5 or with
individuals with known or suspected immune suppression or
active eczema. Pregnant women or those at risk of pregnancy
were also excluded from the trial.
After screening investigations, patients were excluded if
they had any hematological evidence of immunosuppression or
abnormal renal or liver function. Fourteen women were
screened for inclusion in the trial, but 2 were excluded, 1 on the
basis of a CD4 lymphocyte count below the normal limit and the
other because of abnormal liver function tests requiring additional investigation. During the recruitment period, no women
below the age of 42 years were eligible for consideration for
inclusion in the trial.
Twelve women entered the trial and all completed the
treatment and follow-up. Eleven women had VIN, and one had
lower vaginal disease. After vaccination, all subjects were followed for a 6-month period. Trial visits were initially monthly
until 3 months, followed by a final review at 6 months. No other
treatment for the genital intraepithelial neoplasia was given
during the trial or in the month preceding the vaccination.
The study was approved by the local research ethics committee of the hospital, the local genetic manipulation committee,
the national Gene Therapy Advisory Committee, the National
Health and Safety Executive, and the Medicines Control
Agency.
Vaccination
The recombinant vaccinia virus expressing HPV-16 and
HPV-18 E6 and E7 (TA-HPV) has been described previously
(20, 24, 25). It consists of the fused E6 and E7 open reading
frames of HPV-16 and HPV-18, each under the control of a
vaccinia promoter within the Wyeth strain of vaccinia virus. The
E7 gene in both cases was modified by mutation of the retinoblastoma gene product binding sequence so that there can be no
binding of the resultant viral protein with retinoblastoma protein. The fused E6 and modified E7 genes have previously been
demonstrated to show no transforming activity. The virus was
prepared at a concentration of 1 ⫻ 108 plaque-forming units/ml
as used previously and known to be able to stimulate an immune
response (20, 24, 25).
TA-HPV (in an estimated dose of 2.5 ⫻ 105 plaqueforming units) was introduced into the patient by scarification
through a 50 ␮l drop of the virus suspension applied to the skin
of the upper arm overlying the deltoid muscle. The area was
allowed to dry before covering with a waterproof dressing
(Opsite Plus, Smith & Nephew). The dressing was changed
twice a week for ⬃4 weeks until the scab, which formed as a
result of the virus-induced inflammation, had separated and the
vaccination site was healed. Samples for virological detection
were taken from the surface of the dressing every week before
its removal to test for the risk of possible contamination of the
environment, and on separation, the final scab was tested for the
presence of live virus.
HPV Typing
Biopsies from subjects were immediately snap frozen in
liquid nitrogen. DNA was subsequently extracted by proteinase
K digestion overnight in an extraction mix (comprising 200
␮g/ml proteinase K, 0.5% SDS, 10 mM Tris, and 10 mM EDTA
at pH 7.5). PCR using primers directed at a 268-base region of
the ␤-globin gene (26) was used to assess the adequacy of the
sample DNA for HPV typing. Typing was then performed by
PCR with the GP5⫹/GP6⫹ primer pair (27) to amplify a region
within the L1 gene of genital HPV types. The DNA amplification reaction was performed in 50 ␮l using 2.5 ␮l of a hot-start
Taq polymerase (HotStarTaq; Qiagen) and the PCR product
purified away from the other reaction reagents using a PCR
purification kit (Qiagen). The HPV PCR product was sequenced
directly and the sequence compared with the L1 open reading
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Clinical Cancer Research 5207
frame of all known genital HPV sequences available in the HPV
database (Los Alamos National Laboratory, University of California).3
Antivaccinia ELISA
Whole blood was collected in one 7.5-ml S-Monovette tube
(Sarstedt 01.1601.001) at days 0, 28, 56, and 84. Serum was
separated and stored at ⫺20°C until required for analysis. Vaccinia-specific IgG was measured in patient sera by ELISA using
Wyeth strain vaccinia-infected Vero cell lysates as antigen and
mock-infected Vero cell lysates as the control (20). The titer at
absorbance 0.5 was determined using the line of best fit from the
graph of the log sample dilution versus sample Wyeth absorbance minus sample Vero absorbance.
ELISPOT Assays
Whole blood was collected in S-Monovette 8.5 ml of citrate
phosphate dextrose adenine tubes (Starstedt 01.1610.001), and
PBMCs were isolated by density centrifugation on the same day.
Cells were cryopreserved in vapor phase nitrogen until required.
Analysis of HPV-16-specific T-Cell Responses with
Overlapping E6 and E7 Peptides. IFN-␥ ELISPOT assays
were performed as described previously (23). Briefly, PBMCs
were thawed and seeded at a density of 2 ⫻ 106 cells/well of a
24-well plate (Costar, Cambridge, MA) in 1 ml of Iscove’s
medium (Life Technologies, Inc.) enriched with 10% human
antibody serum, in the presence or absence of indicated E6 and
E7 peptide pools. As a positive control, PBMCs were cultured in
the presence of a memory recall mix, consisting of a mixture of
tetanus toxoid (0.75 flocculation units/ml final concentration;
National Institute of Public Health and Environment, Bilthoven,
the Netherlands), Mycobacterium tuberculosis sonicate (2.5
g/ml; generously donated by Dr. P. Klatser; Royal Tropical
Institute, Amsterdam, the Netherlands) and Candida albicans
(0.005%; HAL Allergenen Lab, Haarlem, the Netherlands). The
peptides used spanned the HPV-16 E6 and E7 protein and
consisted of 15 E6 and 9 E7 overlapping 22-mer peptides.
Peptides were used in pools of four to five peptides at a concentration of 5 g/ml/peptide. The peptides, as indicated by their
first and last amino acid, were used in the following pools: E6-I,
1–22, 11–32, 21– 42, and 31–52; E6-II: 41– 62, 51–72, 61– 82,
and 71–92; E6-III, 81–102, 91–112, 101–122, and 111–132;
E6-IV, 111–132, 121–142, 131–152, and 137–158; E7-I, 1–22,
11–32, 21– 42, and 31–52; and E7-II, 41– 62, 51–72, 61– 82,
71–92, and 77–98 (23). After 4 days of incubation at 37°C,
PBMCs were harvested washed and seeded in four replicate
wells at a density of 105 cells/well in 100 ␮l of Iscove’s medium
(Life Technologies, Inc.) enriched with 10% FCS in a multiscreen 96-well plate (Millipore, Etten-Leur, the Netherlands)
coated with an IFN-␥-catching antibody (Mabtech AB, Nacha,
Sweden). The ELISPOT was otherwise performed according to
the instructions of the manufacturer (Mabtech AB). The number
of spots was analyzed with a fully automated computer-assisted
video imaging analysis system (Carl Zeiss Vision). The background number of spots in the medium controls was 2.3 ⫾ 2
3
Internet address: http://hpv-web.lanl.gov/stdgen/virus/hpv/.
spots/50,000 PBMCs. Specific spots were calculated by subtracting the mean number of spots ⫹2⫻ SD of the medium only
control from the mean number of spots in experimental wells.
Antigen-specific T-cell frequencies were considered to be increased compared with nonresponders when specific T-cell frequencies were ⬎1/10,000 (23). T-cell frequencies were considered to be boosted by the vaccine when they were at least 3-fold
higher than before vaccination. In our experience, the use of
these long peptides, which need to be taken up, processed and
presented by antigen-presenting cells, together with the long
incubation period, highly favors the response of CD4⫹ cells
(23, 28, 29).
Analysis of CD8ⴙ T-Cell Responses with HLA Class
I-restricted HPV Epitopes. ELISPOT plates (MAIPS410;
Millipore) were prepared with the following antigens in triplicate wells: (PHA) 1 ␮g/ml; synthetic peptides (Immune Systems
Ltd.) of HPV-16 E629 –38, 59 – 67, 80 – 88 and E711–20, 82–90, 86 –93
(30, 31) influenza virus M158 – 66; and EBV BMLF1280 –288 all at
25 ␮g/ml or inactivated vaccinia virus and control-uninfected
Vero cell lysate 1:2500. Patient PBMCs from each time point
were thawed from cryopreserved stocks and resuspended in
medium [RPMI (Life Technologies, Inc.), 10% antibody serum
(Sigma), 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 2 mM
6
L-glutamine (all Life Technologies, Inc.] at 5 ⫻ 10 /ml. PBMCs
were then added to wells in triplicate, 2.5 ⫻ 105 to the PHA
wells and 5 ⫻ 105 to each of the remaining wells containing
antigen or medium alone. After overnight incubation and washing, biotinylated monoclonal antibody to IFN-␥ (7-B6-1-biotin;
Mabtech) was added, followed by streptavidin-conjugated
horseradish peroxidase (Mabtech). The assay was developed
according to the manufacturer’s instructions (Mabtech). A positive response to antigen was defined as ⬎20 spots (IFN-␥producing cells)/106 PBMCs in response to antigen (after subtraction of background). A positive response after vaccination
was recorded if the frequency of IFN-␥-producing cells postvaccination was greater than twice the prevaccination response
to an antigen. In our experience, the use of well-defined minimal
CTL peptide-epitopes results in strong responses of CD8⫹ T
cells, whereas CD4⫹ T-cell reactivity is usually weak or not
detectable because of the short incubation time (23).
Clinical Response
The extent of the disease was assessed by direct measurement and photographic record of up to two marker lesions in
each case. The longest diameter of each marker lesion was
recorded serially through the study. Three biopsies of the visible
areas were taken at time points: before the vaccination and at 12
and 24 weeks after vaccination. Care was taken to ensure that
the biopsies were taken so as not to directly influence the
diameter of the lesion being studied. The biopsies were analyzed
by two independent pathologists (R. P. M. and N. C.) and the
intraepithelial neoplasia graded according to the three-tier International Society for the Study of Vulvovaginal Disease classification. The presence of HPV detectable by polymerase chain
amplification in tissue was sought at each biopsy point. Any
symptomatic change was recorded with particular reference to
irritation and pain. Assessment was by subjective grading of
symptoms into one of four categories: nil; mild; moderate; or
severe. After vaccination, subjects were actively questioned to
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5208 Therapeutic HPV-16 E6/E7 Vaccination for VIN/VAIN
Table 1
Patient study no.
Age (yr)
Disease
1
2
3
4
5
6
7
8
9
10
11
12
48
49
47
49
44
47
42
49
45
42
52
53
VIN 3
VIN 3
VIN 3
VIN 3
VAIN 2
VIN 3
VIN 3
VIN 3
VIN 3
VIN 3
VIN 3
VIN 3
Patients enrolled in study
Duration of disease
11 years
8 years
1 year
2.5 years
3 months
6 months
1 year
2 years
3 months
4 months
14 years
3 months
HPV type
HLA A type
16
16
16
16
16
16
16
16
16
33
16
16
2, 68
2, 3
2, 24
2, 3
2, 3
1, 2
2, 32
1, 24
2, 26
2, 24
2
3, 3
ascertain the nature of any systemic symptoms potentially attributable to vaccination, and all additional medications were
recorded.
RESULTS
Patients and HPV Type
Twelve women were recruited for vaccination (Table 1).
The age range was 42–54 years with a mean of 46 years. The
diagnosis of AGIN had been made between 3 months and 14
years (mean, 3.4 years) before the start of the vaccine trial. The
disease was multifocal in 8 women. Eleven women had VIN
(grade III), and 1 woman (no. 5) had VAIN (grade II). Previous
treatments received by half the patients included excisional
surgery, laser ablation, and topical imiquimod cream. All patients were positive for HR-HPV, with 11 of the 12 women
infected by HPV-16. HPV-33 was identified in 1 woman (no.
10). Nine of the patients were HLA A2 haplotype.
Safety
There was no detectable adverse effect on kidney, liver, or
bone marrow function as assessed by blood samples and in no
patient was there a significant increase in disease area or progression. All patients remained generally well during and after
the vaccination. A local reaction at the site of vaccination at
⬃7–10 days was common but temporarily limited arm movement in only 2 patients. The swabs taken from the outside of the
dressing each week before its removal showed no evidence in
any case of escape of the active virus from beneath the dressing
to the exterior. A total of 57 swabs from 12 patients was
assayed, and no virus particles were detected in any of the
samples.
Occlusive dressings were worn over the vaccination site
until the scab formed, dried and then separated from the skin.
Gauze samples from under each dressing were tested for the
presence of live TA-HPV. Live virus was detected in the under
surface of the dressing in all patients after vaccination for a
mean of 21 days. In 5 of the 12 patients, live virus was detected
in the final sample containing the scab. In 7 patients, no live
virus was detected in the final sample. A small area of scarring
was left at the vaccination site.
Clinical Response
Ten of the 12 treated women (83%) experienced some
reduction in the size of the affected area over the 6 months after
Fig. 1 Percentage reduction in lesion size. Sum of widest diameter(s)
at 6 months shown as a percentage reduction compared with the diameter prevaccination.
vaccination with a mean reduction in marker lesion diameter of
40% (Fig. 1). In 1 woman (no. 5), there was complete clearance
of the abnormal area with histological normality at 3 months
after vaccination. In 9 women, the grade of the marker lesion(s)
remained unchanged, but there was a reduction of the extent of
the abnormal epithelium. The size of the affected area was
assessed by the longest diameter of identifiable lesion(s). In
these 9 individuals, the reduction in length ranged from 22 to
65% with 5 of the treated women showing at least 50% reduction in total maximum lesional diameter. Two women (nos. 1
and 10) showed no reduction in the size of the affected area of
vulval disease.
In the 11 women in whom VIN was still present at the end
of the study, the same HPV was still detectable in the abnormal
epithelium. In 1 patient (no. 5) whose VAIN cleared after
vaccination, HPV-16 was no longer detectable in the previously
abnormal area of the vagina.
The symptoms of VIN experienced by the patients in the
study included irritation, soreness, and pain. There was no
overall change in symptom score between pre- and postvaccination, with 3 women noticing a slight worsening of symptoms,
3 women having a slight improvement, and 6 women experiencing no change.
Immune Response
Vaccine-Induced Immunity against the Vaccinia Vector. To monitor the impact of the vaccine on the immune
system, the responses to vaccinia were measured. Both antibody
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Clinical Cancer Research 5209
Fig. 2 Vaccination induced immunity
against the vaccinia vector. Pre- and postvaccination antibody and T-cell response
above background to the vaccinia vector
were detected in patient serum and PBMC
samples, respectively, taken before vaccination (day 0) and at days 28, 56, and 84
after vaccination. Peak serum antibody
(IgG) responses were measured by ELISA
prevaccination (Œ) and postvaccination
(䡺). Frequency of IFN-␥-producing T
cells responding to vaccinia vector was
measured by ELISPOT in patient PBMC
prevaccination ( ) and postvaccination ( ).
In each case, the maximum response postvaccination is shown.
and T-cell responses against vaccinia were found to be strongly
increased after vaccination in almost all patients (Fig. 2). Before
vaccination antivaccinia IgG was detected in 9 of 12 patients
(nos. 2, 3, 4, 5, 6, 8, 9, 10, and 12). Postvaccination IgG
responses in 11 of 12 patients (not no. 8) increased ⬎10-fold.
Prevaccination T-cell responses to the Wyeth strain vaccinia
virus as measured by ELISPOT were positive in 6 of 12 patients
(nos. 2, 4, 5, 6, 9, and 10), with frequencies ranging from
1:13,000 to 50,000. After vaccination, the IFN-␥-producing
T-cell frequency to the vaccinia vector increased by ⬎2-fold
(frequencies 1:2,000 –10,000) compared with prevaccination
levels in all patients, except no. 4. No positive IFN-␥ responses
were detected against the Vero lysate control. The increase in
antivaccinia responses postvaccination indicated that the patients were able to respond to the vaccinia vector component of
the vaccine TA-HPV after vaccination.
Prevaccination HPV 16-Specific Immune Responses.
Preexisting HPV-16-specific immunity was detected in 6 of the
10 patients (nos. 2, 4, 5, 7, 10, and 12; Table 2), all of whom
showed reactivity within the HPV-16 E6 protein. Three subjects
(nos. 4, 5 and 7) also demonstrated HPV-16 E7-specific T-cells.
Vaccine-Induced HPV-16-Specific Immune Responses.
TA-HPV-induced HPV16 E6 and E7-specific T-cell immunity
was analyzed by the stimulation of PBMCs with a set of overlapping peptides covering the complete amino acid sequence of
the E6 and E7 proteins. From 10 of 12 patients, enough material
was available to study the HPV-16-specific immune response
before vaccination as well as 4 and/or 8 weeks after vaccination
with TA-HPV (Table 2). Upon vaccination, 4 patients (nos. 1, 4,
6, and 10) did not mount an increased response to the HPV-16
peptide pools. Two of these patients (nos. 1 and 10) also showed
no clinical reduction in the size of the lesion(s). Importantly,
HPV-16 E6- and E7-specific T cells were specifically enhanced
after vaccination in 6 of the 10 patients tested (nos. 2, 3, 5, 7, 11,
and 12; Table 2 and Fig. 3). In 1 case (no. 3) the T-cell response
was focused to one peptide pool, whereas in the remainder (nos.
2, 5, 7, 11, and 12), T-cell reactivity was detected against two or
three different peptide pools. All patients reacted to the positive
memory recall mix.
In addition, PBMCs were stimulated with six CTL epitopes
identified within HPV-16 E6/E7 and chosen for their ability to
bind MHC class I molecules of HLA-A*0201-positive individuals (30, 31). Of the 10 patients typed as HLA-A2 (Table 1), 1
patient (no. 3) responded weakly to peptide HPV-16 E680 – 88
(T-cell frequency 1/50,000). This response was already present
before vaccination and the single injection of TA-HPV failed to
enhance this reactivity. Two patients (nos. 7 and 12) showed an
enhanced production of IFN-␥ in response to the whole HPV-16
E6 and E7 protein as expressed in TA-CIN after vaccination
with TA-HPV. No other patient reacted to TA-CIN either pre- or
postvaccination.
DISCUSSION
We have analyzed the safety and immunogenicity of the
recombinant vaccinia vector expressing HPV-16 and HPV-18
E6 and E7 proteins (TA-HPV) in a Phase II clinical trial. In this
study of 12 women with high-grade VIN and VAIN, no adverse
effects of the vaccination were found. In general, the patients
felt well without definite evidence of pyrexia. The vaccination
site was tender and uncomfortable in all women with duration of
discomfort ranging between 1 and 13 days, which was maximal
⬃1 week after vaccination. The vaccination site was dressed for
a mean duration of 25 days (range, 15–32 days) and was
acceptable to all patients. Vulval symptoms were not significantly improved by vaccination. It may be that an alternative
reporting system, e.g., visual analogue scale might prove more
sensitive in detecting symptom change.
No progression of disease was observed in any of the 12
women during the 6 months after vaccination. In 11 women, no
change in disease grade occurred, but the reduction in size of the
measured regions of AGIN seen in 10 of the 12 women was
promising. In 5 women, the affected areas were reduced by
⬎50% of the original longest diameter, and in 1 patient, the
lesion cleared completely. An effective treatment that would
compare favorably with the most common form of treatment,
excisional surgery, would be one in which complete clearance
occurred and in which recurrence was rare. The responses seen
in this small group of women with diverse duration of disease
and prior treatment do not achieve this ideal, although as yet, it
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5210 Therapeutic HPV-16 E6/E7 Vaccination for VIN/VAIN
Table 2 TA-HPV enhanced HPV-16 E6- and E7-specific immune responses
PBMCs of 10 patients were available for complete analysis of the HPV-16 E6- and E7-specific T-cell response by IFN-␥-ELISPOT assay.
Specific spots/50,000 PBMCs are indicated. Specific spots were calculated by subtracting the mean number of spots ⫹ 2⫻ SD of the medium control
from the mean number of spots of experimental wells. Indicated in bold are those peptide pools (and number of spots) to which specific T-cell
frequencies were ⱖ1/10,000 PBMCs and at least 3-fold the pre vaccination response because these are considered to be vaccine enhanced. NA, not
available for analysis. ⬍1, no specific response to E6 or E7. MRM, memory recall mix used as a positive control.
Patient
Date (weeks)
E6 Pool 1
E6 Pool 2
E6 Pool 3
E6 Pool 4
E7 Pool 1
E7 Pool 2
MRM
1
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
0
4
8
⬍1
1
NA
⬍1
1
⬍1
⬍1
⬍1
5
⬍1
NA
⬍1
⬍1
⬍1
1
⬍1
⬍1
⬍1
4
23
15
⬍1
⬍1
⬍1
1
⬍1
⬍1
4
84
39
⬍1
1
NA
26
1
26
2
57
35
6
NA
⬍1
5
18
86
⬍1
⬍1
1
21
84
66
⬍1
0
⬍1
⬍1
⬍1
⬍1
5
1
⬍1
1
4
NA
⬍1
3
⬍1
⬍1
⬍1
⬍1
⬍1
NA
⬍1
1
9
15
⬍1
⬍1
⬍1
7
1
⬍1
1
2
⬍1
1
⬍1
⬍1
13
42
17
⬍1
⬍1
NA
⬍1
5
27
⬍1
⬍1
⬍1
⬍1
NA
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
1
7
15
7
6
⬍1
⬍1
16
2
⬍1
⬍1
⬍1
1
NA
⬍1
⬍1
7
⬍1
2
⬍1
⬍1
NA
⬍1
10
10
1
⬍1
⬍1
4
5
44
24
1
1
⬍1
3
9
⬍1
4
⬍1
⬍1
⬍1
2
NA
⬍1
1
⬍1
⬍1
⬍1
⬍1
6
NA
⬍1
3
14
33
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
1
⬍1
⬍1
⬍1
⬍1
4
⬍1
⬍1
23
49
NA
100
100
100
50
46
57
124
NA
103
119
87
85
61
79
63
114
106
91
51
108
79
63
93
54
104
58
71
2
3
4
5
6
7
10
11
12
is impossible to know if the longer term course of the disease
has been influenced by the stimulation of anti-HPV immunity.
Interestingly, the patient who showed a complete response (no.
5) also showed resolution of concurrent grade I CIN. Although
spontaneous resolution of low-grade cervical disease is common
(32), persistence of her clinical response to 24 months after
vaccination is extremely encouraging, especially given her prolonged (⬎10 year) pretrial history of relapsing cervical and
latterly vaginal disease. A reduction in lesion size also enabled
several subjects to undergo relatively minor excisional treatment
at the conclusion of the study, compared with the radical approach that might have been used to deal with their disease at
enrolment.
It has earlier been reported that women with HPV-16
cervical disease may have T-cell responses to HPV-16 E7
peptides (23, 33, 34). In our patients, we found measurable
immune responses to at least one HPV-16 E6 or E7 peptide pool
in 6 of the analyzed women (60%) prevaccination (Table 2).
Immunoreactivity was found against all HPV-16 E6 and E7
peptide pools but with slightly greater frequency or strength to
peptides covering the central E6 region from amino acid 41-132.
Others have suggested that T-cell responses to HPV-16 E7
peptides may be reduced in women with invasive cancer (23,
35) but relatively increased in persistent and especially high-
grade cervical dysplasia (35). The low level of HPV-16 E7stimulated IFN-␥ production by T cells from this cohort of
patients with VIN 3 could reflect an apparent suppression of
immunity in chronic HPV disease or tolerance to 16 E7. Previously reported preponderance of immunoreactivity to COOHterminal HPV-16 E7 peptides in patients with CIN (34, 35) or
central E7 peptides in high-grade CIN or cervical cancer (23)
was not seen in these patients with VIN/VAIN.
A systemic immune response upon vaccination was detected by ELISPOT assays to pooled peptides in 6 of the 10
patient samples available for testing (Table 2 and Fig. 3). All
these 6 subjects showed a clinical response. In 4 women (40%),
there was no significant increase in the number of lymphocytes
producing IFN-␥ after exposure to viral peptides after vaccination compared with the prevaccination responses. Two of these
4 women had no clinical response either, but the remaining 2
patients showed a significant (⬎50%) reduction in marker lesion diameter. There was no strong correlation between the
clinical change and the observed in vitro T-cell response. It is
possible, however, that the detection of systemic immunity to
HPV-16 E6 and E7 peptides after vaccination may not truly
reflect the intensity of the immune response at the site of the
disease. Nevertheless, the observed T-cell responses in 6 of 8
clinical responders remain remarkable.
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Clinical Cancer Research 5211
Fig. 3 TA-HPV induced T-cell responses in patient nos. 3, 7, and 10.
Three examples, representing a two responders (nos. 3 and 7) and a
nonresponder (no. 10), are shown at day 0 (prevaccination), day 28 (4
weeks after vaccination), and day 56 (8 weeks after vaccination). TAHPV induces HPV-16 E6- and E7-specific IFN-␥ responses. The mean
number of spots and SE bars/50,000 PBMCs induced by the medium
control or the peptides present in the E6 and E7 peptide pools are
depicted. As positive control a memory recall antigen mix (see “Materials and Methods”) was used.
Other studies of vaccination as therapy for HR-HPV-associated anogenital disease have been reported recently. The use
of DNA (35), peptides (21–23, 36, 37), protein (38), recombinant virus (20), dendritic cells (39), and virus-like particles (40)
have all been shown to induce an HPV-specific immune response. The development of a cytotoxic T-cell response probably in combination with a T-helper cell response is likely to
produce the best immune attack on virally infected neoplastic
cells. HLA class I-restricted HPV-16 E7 peptides most likely to
produce a cytotoxic T-cell response have been defined (30, 31),
but in our patients, there was no stimulation of T-cell response
to the peptides after vaccination. However, the use of a restricted set of peptides to analyze CTL reactivity may not permit
the detection of all CTL responses. Other studies of vaccination
in humans using these selected peptides have suggested that
patients with CIN, cervical cancer, and vaginal cancer are able
to mount a CD8⫹ T-cell response to the peptides (22, 36),
although in one study, the lack of a peptide-specific cytotoxic
T-cell response was similar to the results presented here (37). In
contrast, the responses to class II-restricted epitopes of HPV-16
E7 (23) was stimulated after vaccination in 6 of 10 women
tested. These responses have not been tested in other vaccination
trials in humans.
The effect of vaccination on HPV infection and HPVassociated neoplastic disease is less clear. Only small numbers
of patients with HR-HPV dysplasia or carcinoma have received
vaccination as therapy, and as yet, dramatic clinical responses
have not occurred. Borysiewicz et al. (20) reported the use of
TA-HPV in 8 women with therapy-unresponsive cervical cancer, 1 of whose disease remitted. Although the vaccine has been
used as adjuvant therapy in two small trials of women with CIN
3 before laser therapy and in women with early-stage cervical
cancer before surgery, the possible effect on the disease was not
evaluated (41). HPV peptides have been used as therapeutic
vaccines in several small studies. In one of these, 11 women
with cervical cancer and 1 woman with vaginal cancer, all of
whom were of the HLA-A2 genotype, received immunotherapy
with the HPV-16 E786 –93 peptide (36). None of these patients
showed a clinical response to vaccination, although 2 individuals demonstrated induction of specific and persistent CTL responses. Another HLA-A2-restricted peptide E712–20 has been
tested as a therapeutic vaccine in 18 women with HPV-16positive high-grade intraepithelial neoplasia of the cervix (n ⫽
16) or vulva (n ⫽ 2; Ref. 22). Seven of these women also
received the same HPV-16 E7 peptide, E786 –93 used by Steller
et al. (36). There was observed improvement of disease in terms
of partial (6 of 18) or complete (3 of 18) clearance of affected
areas with improvement only occurring in women with CIN.
Ten of 16 women tested had induction of E7 immunoreactivity
as assessed by IFN-␥ release and chromium release assays. The
combination of these two HPV-16 E7 peptides, E711–20 and
HPV-E786 –93, has also been tested as therapy in 19 women with
late-stage HPV-16-positive cervical cancer who were HLAA*0201-positive (21). The vaccination was well tolerated but
produced no clear effect on disease course. A third type of
vaccine tested in human disease has recently been reported (35).
Plasmid DNA encoding the amino acids 83–95 of HPV-16 E7
was used as a vaccine in patients with AIN. Three of 19 treated
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Research.
5212 Therapeutic HPV-16 E6/E7 Vaccination for VIN/VAIN
individuals were found to have changed from high- to low-grade
AIN, whereas in the remainder, the disease did not change.
Ideally, therapeutic vaccination for HPV-associated AGIN
would have an efficacy that induces complete disease regression
without recurrence. To produce such an effect, the immune
response should be readily induced and maintained and must be
able to produce a potent cytotoxic or delayed type hypersensitivity response against epithelial cells harboring the viral DNA.
The development of a therapeutic vaccine to combat this virally
driven disease is still some way from producing these aims.
However, this trial and others suggest that stimulation of the
immune response in HR-HPV-infected individuals may be able
to produce at least a partial effect on neoplastic disease. Improvements in vaccine design and delivery as well as techniques
to optimize immune responses within the diseased epithelium
may ensure that therapeutic vaccination becomes an effective
treatment choice for this group of conditions.
ACKNOWLEDGMENTS
We thank the staff of the Colposcopy Clinic, Vulval Clinic, and the
Clinical Investigation Ward at Addenbrooke’s National Health Service
Trust for their help with the clinical aspects of this study and all past and
present members of the TA-HPV project team at Xenova Research Ltd.
We also thank those women who kindly agreed to participate in this
Phase II trial.
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Vaccinia-Expressed Human Papillomavirus 16 and 18 E6 and
E7 as a Therapeutic Vaccination for Vulval and Vaginal
Intraepithelial Neoplasia
Peter J. Baldwin, Sjoerd H. van der Burg, Christopher M. Boswell, et al.
Clin Cancer Res 2003;9:5205-5213.
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