Abstract Human papillomavirus (HPV) infection can cause genital warts and cervical cancer. HPV types 6 and 11 cause &gt;90% of genital wart cases; HPV16 and 18 cause 70% of cervical cancers. A prophylactic HPV (types 6, 11, 16, 18) L1 virus-like particle (VLP) vaccine may substantially reduce the incidence of these lesions. This report describes the results of a phase I study of the HPV18 component of such a vaccine. Forty women were randomized to receive either HPV18 L1 VLP vaccine or placebo. Anti-HPV18 responses were measured using a competitive radioimmunoassay (cRIA). Tolerability was evaluated using vaccination report cards (VRC). The study showed that the HPV18 L1 VLP vaccine was generally well-tolerated and highly immunogenic. Peak anti-HPV18 geometric mean titers (GMT) in vaccines were 60-fold greater than those observed in women following natural HPV18 infection. Further studies of a multivalent HPV L1 VLP vaccines are warranted

Anna R Giuliano

Deemarie Skulsky

Eliav Barr

Frank J Taddeo

Gretchen Tamms

Judith F Smith

Kathrin U Jansen

Kevin A Ault

Lee-Lian Kim

Maria Allende

Robert P Edwards

CiteSeerX

Vaccine 22 (2004) 3004–3007
A phase I study to evaluate a human papillomavirus
(HPV) type 18 L1 VLP vaccine
Kevin A. Ault a,∗, Anna R. Giulianob, Robert P. Edwardsc,1, Gretchen Tammsd,
Lee-Lian Kimd, Judith F. Smithd, Kathrin U. Jansend, Maria Allended, Frank J. Taddeod,
DeeMarie Skulskyd, Eliav Barrd
a Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, 200 Hawkins Drive,
Iowa City, IA 52242, USA
b Arizona Cancer Center, University of Arizona, Tucson, AZ, USA
c Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA
d Merck Research Laboratories, Merck and Co. Inc., West Point, PA, USA
Received 30 October 2003; received in revised form 6 February 2004; accepted 12 February 2004
Available online 17 March 2004
Abstract
Human papillomavirus (HPV) infection can cause genital warts and cervical cancer. HPV types 6 and 11 cause >90% of genital wart
cases; HPV16 and 18 cause 70% of cervical cancers. A prophylactic HPV (types 6, 11, 16, 18) L1 virus-like particle (VLP) vaccine may
substantially reduce the incidence of these lesions. This report describes the results of a phase I study of the HPV18 component of such
a vaccine. Forty women were randomized to receive either HPV18 L1 VLP vaccine or placebo. Anti-HPV18 responses were measured
using a competitive radioimmunoassay (cRIA). Tolerability was evaluated using vaccination report cards (VRC). The study showed that
the HPV18 L1 VLP vaccine was generally well-tolerated and highly immunogenic. Peak anti-HPV18 geometric mean titers (GMT) in
vaccines were 60-fold greater than those observed in women following natural HPV18 infection. Further studies of a multivalent HPV L1
VLP vaccines are warranted.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Human papilloma virus; Vaccination; Serology
1. Introduction
Human papilloma virus (HPV) infects >50% of
sexually-active adults[1] and is responsible for genital
warts and cervical intraepithelial neoplasia (CIN), an ab-
normality that may lead to cancer. Four HPV types have
been associated with the majority of these diseases: HPV6
and 11 cause >90% of genital wart cases, HPV16 causes
50% of cervical cancer cases, and HPV18 causes 20% of
cervical cancer cases. HPV18-related cervical cancers are
associated with especially high mortality, because they of-
ten present as adenocarcinomas[2], lesions that are poorly
detected by Pap testing and are difficult to treat[3].
 These data were presented, in part, at the 14th meeting of the Inter-
national Society for STD Research in Berlin, Germany on 27 June 2001.
∗ Corresponding author. Tel.:+1-319-353-7384; fax:+1-319-356-3901.
E-mail address: kevin-ault@uiowa.edu (K.A. Ault).
1 Present address. Department of Obstetrics and Gynecology, University
of Louisville, AY 300550 South Jackson Street, Louisville, KY 40202,
USA.
Vaccination against common HPV types may reduce
the burden of HPV-related diseases. HPV L1 capsid pro-
teins produced in modified yeast self-assemble into empty
virus-like particles (VLPs) that resemble the wild-type
virion but are non-infectious. In several early phase clinical
studies, HPV11 and 16 L1 VLP vaccines were observed
to be generally well-tolerated and to induce substantial
anti-HPV11 and 16 responses, respectively[4–6]. In two
recent studies, administration of an HPV16 L1 VLP vaccine
was shown to prevent HPV16 infection and related CIN in
100% of HPV16-näıve vaccines[5,6].
Because administration of HPV L1 VLP vaccine is likely
to result in type-specific immunity, prophylactic vaccines
that target the majority of cancer-causing HPV types should
include both HPV16 and HPV18 components. Here we de-
scribe a phase I clinical trial that investigated the immuno-
genicity and tolerability of the HPV18 L1 VLP component
of such a vaccine.
0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2004.02.020
K.A. Ault et al. / Vaccine 22 (2004) 3004–3007 3005
2. Methods
A double-blind, randomized, placebo-controlled phase
I study of an HPV18 L1 VLP vaccine (Merck Research
Laboratories, West Point, PA) was conducted in 40,
16–23-year-old women recruited from three US college
campuses. Potential participants were excluded if they
reported a prior abnormal Pap smear or more than five life-
time male sexual partners. Additionally, women were not
required to be HPV18-seronegative or PCR negative upon
enrolment.
Volunteers were randomized in a 2:1 vaccine to placebo
ratio. Each dose of HPV18 L1 VLP vaccine consisted of
80g of HPV18 L1 VLP formulated with 450g of amor-
phous aluminum hydroxyphosphate sulfate adjuvant in a
buffered solution yielding a total injection volume of 0.5 ml.
The 80g dose, the highest dose contemplated for Merck’s
investigational quadrivalent HPV (types 6, 11, 16, 18) L1
VLP vaccine, was selected because the current study was
intended to study the vaccine’s tolerability. Placebo vacci-
nation consisted of the aluminum adjuvant and buffered so-
lution only. Participants were vaccinated at day 0, months 2,
and 6. The vaccine and placebo were visually indistinguish-
able. Participants were required to use effective contracep-
tion during the trial.
At day 0 and month 7, participants underwent physi-
cal examination, genital sampling for HPV, and Pap testing
(ThinPrepTM, Cytyc, Boxborough, MA).
Swabs collected from the cervix, vagina, labia, vulva, per-
ineum, and perianal area were tested for the presence of
HPV18 DNA using a polymerase chain reaction (PCR) as-
say targeting the HPV18 L1, E6 and E7 genes as previously
described[5]. Any sample testing positive for two or three
of these genes was considered positive. Serum samples were
collected at day 0 and months 2, 3, 6, and 7 to measure
anti-HPV18 responses using a competitive radioimmunoas-
say (cRIA). This assay is similar to a previously published
anti-HPV16 VLP cRIA[5]. These assays are based on the
competition of a HPV type-specific, neutralizing monoclonal
antibody with test sera for a limited amount of binding sites
presented by HPV L1 VLPs coated onto beads. Bound mon-
oclonal antibody is detected with125I-anti-mAb. Antibody
titers were determined relative to a non-human primate refer-
ence serum generated by immunization with HPV18 L1 VLP
and were expressed in arbitrary units (milliMerck units/ml,
mMU/ml). An acceptable anti-HPV18 response was defined
as an anti-HPV18 level≥200 mMU/ml 1-month after com-
pletion of the 3-dose vaccination regimen. In one clinical
study with a prototype HPV11 VLP vaccine, anti-HPV lev-
els have been shown to correlate with the capacity to neu-
tralize live HPV virions and with long-term seropositivity
[7].
To measure tolerability, participants completed vaccina-
tion report cards (VRC) after each vaccination. The VRCs
asked participants to report injection site or systemic com-
plaints that occurred during the 14 days following vaccina-
tion. The VRC also required participants to record their oral
temperature 4 h after vaccination, and daily for each of the
ensuing 4 days. Adverse experiences (AEs) were defined as
new physical abnormalities or worsening of pre-existing ab-
normalities that occurred during the 14 days following vac-
cination. Participants were asked to categorize the intensity
of each adverse experience using pre-specified criteria.
An immune response resulting from an ongoing or an-
tecedent HPV18 infection may magnify vaccine-induced
anti-HPV18 responses. Thus, the primary evaluation of
immunogenicity was performed in women who were
HPV18-näıve at enrollment (HPV18 PCR- and seronega-
tive on day 0) and who had not acquired HPV18 infection
during the study (HPV18 PCR negative at month 7). The
protocol pre-specified that the primary analysis would be
conducted in women who had received all 3 doses of study
vaccine, had undergone serologic testing at month 7, and
had not received immunosuppressive medications or blood
products during the course of the study.
The primary immunogenicity analysis was performed
at month 7. Geometric mean titers (GMTs) and the per-
centage of participants who achieved an anti-HPV18 level
≥200 mMU/ml were computed. Since the goal of the study
was to evaluate whether or not the vaccine would induce
anti-HPV18 levels≥200 mMU/ml in greater than half of
the vaccinated subjects, the percentage of subjects with
anti-HPV 18 ≥ 200 mMU/ml was computed separately
among HPV18 L1 VLP vaccine and placebo recipients and
compared to a reference of 50% using exact binomial testing
for a single proportion. Two-sidedP values less than 0.05
were considered statistically significant. Immunogenicity
also was evaluated postdose 2 (month 3).
3. Results
Key demographic characteristics were generally com-
parable between treatment groups. The mean age of par-
ticipants was 20.7 years. Few participants reported prior
gynecologic infections: two participants in the placebo
group had previous occurrences of genital warts, one in the
placebo group had bacterial vaginosis, and two (one each
in the vaccine and placebo groups) had Chlamydia infec-
tion. The median number of lifetime male sexual partners
was 2.0.
A total of 22 women in the vaccine group and 11 women
in the placebo group were included in the per-protocol im-
munogenicity evaluation. Of the seven women who were ex-
cluded, three failed to complete the protocol (one was lost
to follow-up, one withdrew consent, and one experienced
hives and was withdrawn from the study), two were HPV18
PCR positive at enrollment and at month 7, one received
a blood transfusion for gastrointestinal bleeding, and one
was excluded because her month 7 serum sample was not
obtained within the pre-specified time period following the
third vaccination.
3006 K.A. Ault et al. / Vaccine 22 (2004) 3004–3007
0 2 3 6 7
Month
1
13
10
100
1000
2000
3000
G
eo
m
et
ri
c 
M
ea
n 
T
ite
r 
(m
M
U
/m
L
)
HPV 18 L1 VLP Vaccine (80 mcg) (N = 22)
Placebo (N = 11)
Assay Cut-off
Fig. 1. Serological responses to HPV18 L1 VLP expressed as anti-HPV18 cRIA GMTs in mMU/ml on a logarithmic scale with 95% tile confidence
intervals. A GMT of 6.5 mMU/ml indicates a titer below the assay cutoff of 13 mMU/ml.
In the per-protocol cohort, all of the women in the vaccine
group and none in the placebo group developed anti-HPV18
responses 1-month postdose 2 and 3. Serological responses
are summarized inFig. 1. Responders are women who se-
roconverted (above assay cutoff of 13 mMU/ml) follow-
ing the administration of either the HPV18 L1 VLP vac-
cine or placebo. Of the vaccine recipients, 86 and 100%
achieved anti-HPV18 levels≥200 mMU/ml at months 3 and
7, respectively. The proportion of subjects with anti-HPV
18 ≥ 200 mMU/ml was significantly greater than 50% (P <
0.001) at both months 3 and 7. Anti-HPV18 responses in-
creased after each dose of vaccine.
Adverse experiences 0–14 days following any dose
were common among both vaccine and placebo recipients
(Tables 1 and 2). All enrolled women were evaluated for
AEs. Erythema at the injection site appeared more fre-
quently in the vaccine group, (40.7%), compared with the
placebo group, (7.7%), and the difference was nominally
statistically significant (P = 0.035, unadjusted for multiple
Table 1
Local adverse events
Vaccine
(N = 27)
Placebo
(N = 13)
N (%) N (%)
Number (%) of subjects with one or
more adverse site reactions
26 96 11 85
Ecchymosis 1 4 0 0
Erythema 11 41 1 8
Pain, tenderness and/or soreness 26 96 11 85
Pruritis 2 7 0 0
Swelling 10 37 3 23
comparisons). No other statistically significant differences
between placebo and vaccine groups were observed. None
of the injection site AEs were judged by the participants to
be severe in intensity. The most common injection site AE
was pain. Most of the systemic AEs were judged by the par-
ticipants to be mild or moderate in severity. (Table 2) The
proportion of systemic AEs judged to be severe in intensity
were 6.6 and 15.7% in the vaccine and placebo groups, re-
spectively. Systemic AEs are summarized inTable 2. Only
systemic AEs that affected more than 5% of women receiv-
ing vaccine are shown. Headache was the most commonly
reported systemic adverse experience. Systemic AEs were
not different between women receiving vaccine and those
receiving placebo.
Table 2
Systemic adverse events
Vaccine
(N = 27)
Placebo
(N = 13)
N (%) N (%)
Number (%) of subjects with one or
more systemic adverse experience
19 70 11 85
Fatigue 2 7 2 15
Abdominal pain 2 7 1 8
Diarrhea 3 11 1 8
Nausea 2 7 0 0
Headache 13 48 8 62
Upper respiratory infection 2 7 2 15
Pharyngitis 2 7 4 31
Sinus disorder 2 7 0 0
Menstrual disorder 2 7 0 0
K.A. Ault et al. / Vaccine 22 (2004) 3004–3007 3007
4. Discussion
In this study, administration of a 3-dose regimen of
HPV18 L1 VLP vaccine was generally well-tolerated and
induced high titer anti-HPV18 levels in all participants.
In previous studies, administration of HPV16 and
HPV11 L1 VLP vaccines induced substantial peak serum
anti-HPV levels. Brown et al.[7] further demonstrated
that vaccine-induced anti-HPV11 antibodies neutralized a
large input load of live HPV11 virions. Evan et al.[4] and
Harro et al.[6] have reported phase I trials of HPV16 VLP
vaccines. Their findings concerning immunogencity and
tolerability are similar to this report. Finally, two sets of
studies demonstrated that administration of HPV16 L1 VLP
vaccine protected HPV16-naı̈ve women from acquisition
of HPV16 infection[5]. These data suggest that a vaccine
containing HPV18 L1 VLPs may protect against HPV18
infection and related disease. Furthermore, although the
HPV L1 VLP dose used in this study was twice the dose
of VLP 16 L1 used in a previous clinical trial of VLP L1
vaccine[5], the safety profiles were similar.
Although these preliminary studies of HPV L1 VLP
vaccines are encouraging, two questions affecting their ulti-
mate utility remain. First, the minimum protective anti-HPV
level is unknown. In this report, peak vaccine-induced
anti-HPV18 responses were 60-fold higher than anti-HPV18
responses observed in women with detectable serum
anti-HPV18 following clearance of HPV18 infection. The
duration of these antibody responses remains to be deter-
mined.
Second, the impact of prophylactic HPV vaccines on cer-
vical cancer rates remain to be determined. Most HPV in-
fections and low grade CIN clear without sequelae. CIN
2/3 lesions, on the other hand, are the immediate and obli-
gate precursors to cervical cancer. Conservative regimens of
screening and excision of CIN 2/3 lesions have resulted in
large reductions in cervical cancer rates[8,9]. Thus, for use
in cervical cancer prevention, vaccines targeting HPV18 or
other cancer-causing HPV types must demonstrate reduction
in the incidence of CIN 2/3 lesions related to these types.
The ideal prophylactic HPV L1 VLP vaccine should max-
imize coverage of pathogenic HPV types. A vaccine target-
ing HPV 6, 11, 16, and 18 has the potential to impact over
90% of genital wart cases, 50% of CIN 1 cases, and 70%
of CIN 2/3 and cervical cancer cases. Studies of such a vac-
cine are underway. Further evidence supporting a multiva-
lent vaccine has recently been published by Munoz et al.
[10]. A vaccine containing the eight most common onco-
genic HPV types could potentially prevent 95% of cervical
cancers. Thus, an effective and well-tolerated vaccine aimed
at these common HPV types may substantially reduce the
clinical and socioeconomic costs of HPV infection.
Acknowledgements
Informed consent was obtained from all participants in
accordance with US Department of Health and Human Ser-
vices guidelines. The institutional review boards of the Uni-
versity of Iowa, the University of Arizona, and the Univer-
sity of Pittsburgh approved this protocol.
Research funding was provided by Merck and Co. Inc.,
West Point PA USA. Drs. Ault, Giuliano, and Edwards have
received research grants and consultant fees from Merck.
Drs. Allende, Barr, Jansen, Kim, and Taddeo, Ms. Skulsky,
Smith, and Tamms are employees of Merck and Co. Inc.
We are indebted to Tracy Peters of the University of
Iowa, Adrienne Karecki of the Arizona Cancer Center and
Michaleine Bianchi of The University of Pittsburgh for their
expert care of women enrolled in this protocol. Additionally,
Dr. Karen M. Kaplan helped with preparation and revision
of this manuscript.
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A phase I study to evaluate a human papillomavirus (HPV)

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A phase I study to evaluate a human papillomavirus (HPV)

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