Vaccine 24 (2006) 5571–5583
Immunologic responses following administration of a vaccine targeting
human papillomavirus Types 6, 11, 16, and 18
Luisa L. Villa a,∗, Kevin A. Ault b,1, Anna R. Giuliano c,2, Ronaldo L.R. Costa d, Carlos A. Petta e,
Rosires P. Andrade f , Darron R. Brown g , Alex Ferenczy h , Diane M. Harper i , Laura A. Koutsky j ,
Robert J. Kurman k, Matti Lehtinen l, Christian Malm l, Sven-Eric Olsson m, Brigitte M. Ronnett k
, Finn Egil Skjeldestad n , Margareta Steinwall o , Mark H. Stoler p , Cosette M. Wheeler q ,
Frank J. Taddeo r , Jimmy Yu s , Lisa Lupinacci s , Radha Railkar s , Rocio Marchese r ,
Mark T. Esser r , Janine Bryan r , Kathrin U. Jansen r , Heather L. Sings t ,
Gretchen M. Tamms u , Alfred J. Saah u , Eliav Barr u
a
i
Department of Virology, Ludwig Institute for Cancer Research, R. Prof. Antonio Prudente 109, 4th floor, 01509-010 Sao Paulo, SP, Brazil
b Department of Obstetrics, Gynecology and Epidemiology, University of Iowa, Iowa City, IA, USA
c University of Arizona Cancer Center, Tucson, AZ, USA
d Gynecology Department, Instituto Brasileiro de Controle do Cancer, and Hospital do Cancer, Sao Paulo, Brazil
e Department of Obstetrics and Gynecology, Universidade Estadual de Campinas, Campinas Brazil
f CERHFAC-Center of Human Reproduction Studies and Clinical Trials, Curitiba, Brazil
g Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA
h Department of Pathology, McGill University and the SMBD-Jewish General Hospital, Montreal, Que., Canada
Norris Cotton Cancer Center, Departments of Obstetrics & Gynecology and Community & Family Medicine, Dartmouth Medical School, Hanover, NH, USA
j Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA, USA
k Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
l Department of Infectious Disease Epidemiology, National Public Health Institute, Helsinki, Finland
m Karolinska Institute at Danderyd Hospital, Stockholm, Sweden
n Department of Epidemiology, SINTEF Health Research, Trondheim, Norway
o University Hospital, Lund, Sweden
p Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA, USA
q Departments of Molecular Genetics and Microbiology and Obstetrics and Gynecology, University of New Mexico, Albuquerque, NM, USA
r Departments of Vaccine and Biologics Research, Merck Research Laboratories, West Point, PA, USA
s Department of Biostatistics, Merck Research Laboratories, West Point, PA, USA
t Department of Medical Communications, Merck Research Laboratories, West Point, PA, USA
u Department of Biologics Clinical Research, Merck Research Laboratories, West Point, PA, USA
Received 26 November 2005; received in revised form 13 April 2006; accepted 20 April 2006
Available online 15 May 2006
Abstract
Human papillomavirus (HPV) infection causes cervical cancer and genital warts. Young women (1106) were randomized to receive one
of three formulations of a quadrivalent HPV (Types 6/11/16/18) L1 virus-like particle (VLP) vaccine or one of two placebo formulations.
The goal was to assess vaccine safety and immunogenicity in baseline HPV 6/11/16 or 18-naı̈ve and previously infected subjects. All three
∗
1
2
Corresponding author. Tel.: +55 11 3277 6957; fax: +55 11 2189 5036.
E-mail address: [email protected] (L.L. Villa).
Now at Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA, USA.
Now at Moffitt Cancer Center, Tampa, FL, USA.
0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2006.04.068
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L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
formulations were highly immunogenic. At Month 2 (postdose 1), among women with vaccine-type antibodies at baseline, vaccine-induced
anti-HPV responses were ∼12- to 26-fold higher than those observed in baseline-naı̈ve women, suggesting an anamnestic response. Following
an initial, similar sized decline, anti-HPV responses plateaued and remained stable through end-of-study (3.0 years). No vaccine-related serious
adverse experiences were reported.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Human papillomavirus; Vaccine; Immunogenicity
1. Introduction
Human papillomavirus (HPV) infection is a cause of cervical cancer and genital warts [1–6]. HPV infection is also
associated with the development of anal, vulvar, vaginal, and
penile cancers. The oncogenicity of HPV is mediated by the
HPV E6 and E7 proteins, which induce accelerated and disordered cellular proliferation resulting in pre-cancerous epithelial lesions. Such lesions can progress to invasive cancer.
HPV infection is one of the most common sexually transmitted diseases worldwide [5–7]. The lifetime risk of HPV
infection for sexually active males and females is approximately 50% [7]. An estimation of worldwide cancer incidence and mortality for 2002 showed that persistent HPV
infection had caused about 500,000 cases of cervical cancer
[8]. Despite organized screening programs using the Papanicolaou (Pap) test, approximately 35,000 women died from
this disease in the USA and Europe [8]. While Pap testing
reduces mortality from cervical neoplasia, it does not prevent HPV infection or development of pre-cancerous lesions
such as high-grade cervical intraepithelial neoplasia 2/3 (CIN
2/3) and persistent low-grade lesions (CIN 1), all of which
require treatment. A recent study has estimated that in the
United States alone, cervical HPV-related disease accounts
for a total health care cost of $3.4 billion (in 2002 dollars) [9].
Another non-negligible complication of genital HPV infection is anogenital warts, which affect approximately 1% of
sexually active individuals between the ages of 15 and 50
years old [10].
Of the approximately 40 distinct HPV types that are known
to infect the anogenital epithelium, only a small number
cause the majority of disease. HPV Types 16 and 18 cause
approximately 70% of all cervical cancer cases worldwide
[3]. HPV 6 and 11 cause a majority of genital warts (men
and women) [11]. HPV 6, 11, 16 or 18 are found in approximately a third of low-grade cervical lesions [12]. Thus, an
effective HPV (Types 6/11/16/18) vaccine would target HPV
types that cause about 70% of cervical cancers and high-grade
pre-cancerous lesions, about a third of low-grade dysplastic
lesions, and a majority of genital wart cases. Such a vaccine
has the potential to substantially reduce the burden of clinical
HPV disease in both men and women.
HPV is a small, non-enveloped, double-stranded DNA
virus. The HPV L1 capsid protein, when expressed in yeast
cells, forms non-infectious virus-like particles (VLPs) that
resemble native virions [13]. Recent Phase I and II clinical trials have demonstrated that HPV vaccines based on
the HPV L1 capsid protein are generally well-tolerated and
induce neutralizing anti-HPV responses [14–22]. These trials
served as the basis for evaluating a quadrivalent HPV vaccine
targeting HPV 6/11/16/18.
Recently, we described results from a Phase II study
designed to select one of three formulations of quadrivalent
HPV (Types 6/11/16/18) L1 VLP vaccine for use in Phase
III studies. This 3-year, randomized, placebo-controlled trial
showed that the formulation comprising the lowest dose of
HPV 6, 11, 16 and 18 L1 VLPs was generally well-tolerated
and reduced the combined incidence of persistent HPV 6,
11, 16 or 18 infection or related genital disease by 90%, compared to placebo [20]. In this study, subjects with vaccine-type
antibodies or DNA at baseline were eligible for vaccination.
This design feature allowed for an assessment of the tolerability, immunogenicity, and efficacy of the quadrivalent HPV
(Types 6/11/16/18) L1 VLP vaccine in women who had presumably already been infected with vaccine HPV types prior
to enrollment. This report extends previous findings by presenting immunogenicity and tolerability results for all three
doses of quadrivalent vaccine. These data provide the rationale for selecting the quadrivalent HPV vaccine dose used in
Phase III studies. In addition, an exploratory analysis of the
immunogenicity of this Phase III formulation in HPV 6, 11,
16, or 18-naı̈ve and previously infected women, is presented.
2. Methods
2.1. Vaccine
The active quadrivalent vaccine consists of a mixture of
four recombinant HPV type-specific VLPs (Merck Research
Laboratories, West Point, PA) composed of the L1 major
capsid proteins of HPV 6, 11, 16 and 18 synthesized in Saccharomyces cerevisiae [20]. The four VLP types were purified and adsorbed onto a proprietary amorphous aluminum
hydroxyphosphate sulfate adjuvant (AAHS), also referred to
as Merck Aluminum Adjuvant (MAA). The two placebo formulations contained the same adjuvant and were visually
indistinguishable from vaccine.
2.2. Study design
Protocol 007 was a Phase II, randomized, multi-center,
double-blind, placebo-controlled study. The protocol was
designed to select one of three formulations of quadrivalent
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
5573
Fig. 1. Trial profile.
HPV (Types 6/11/16/18) L1 VLP vaccine for use in Phase
III studies and to preliminarily evaluate the safety, immunogenicity, and efficacy of the selected formulation [20]. The
HPV 6, 11, 16 or 18 L1 VLP doses present in each formulation
were based on Phase I HPV 11, 16, and 18 L1 VLP vaccine
safety and immunogenicity studies [16,17,21]. This study
was conducted in two parts (Fig. 1). Part A was a sequential
dose-escalating evaluation. Both the subject and the investigator and his/her staff were blinded to who received vaccine
and who received placebo, but not to the dose of the active
group. Part B was a fully blinded dose-ranging evaluation of
safety, efficacy and immunogenicity. Study procedures for all
subjects in Part A and B were identical. Data presented here
are from Part B, as Part A was not fully blinded.
For Part B, a total of 1106 women with nearly all aged
16–23 years (one subject was 13, other was 15) were recruited
in Brazil, Europe, and the USA. Subjects were randomized in
a 2:2:2:1:1 ratio to receive 20/40/40/20 ␮g of HPV 6, 11, 16
and 18 L1 VLP (including 225 ␮g AAHS), 40/40/40/40 ␮g of
HPV 6, 11, 16 and 18 L1 VLP (including 225 ␮g AAHS), or
80/80/40/80 ␮g of HPV 6, 11, 16 and 18 L1 VLP (including
395 ␮g AAHS), placebo with 225 ␮g of AAHS, or placebo
with 450 ␮g of AAHS. To enrich the study population for
HPV-naı̈ve women, only non-pregnant, healthy women who
reported no prior abnormal Pap smears of low-grade squa-
mous intraepithelial lesion (LSIL) or worse, and reported
a lifetime history of four or fewer male sex partners were
enrolled. Among virgins, enrollment was limited to those
women who were ≥18 years of age and seeking contraception. This study did not exclude subjects with prior or ongoing
HPV infection of any type. Thus, women who were antiHPV seropositive (i.e., had developed immune responses to
an HPV infection) and women who were HPV DNA positive
(i.e., had evidence of ongoing HPV infection) were enrolled.
The study was conducted in conformance with applicable
country or local requirements regarding ethical committee
review, informed consent, and other statutes or regulations
regarding the protection of the rights and welfare of human
subjects participating in biomedical research. All subjects
and/or parents/legal guardians signed informed consents following review of the protocol procedures.
Vaccine or placebo was administered at day 1, Month 2,
and Month 6, given as a 0.5 mL intramuscular injection. All
subjects were observed for at least 30 min after each vaccination for any immediate reaction. Temperatures were recorded
orally for 5 days following each injection. All adverse experiences were collected daily by the participant on a Vaccination
Report Card (VRC) for 14 days following each vaccination.
Participants agreed to use effective contraception during the
vaccination phase.
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L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
A complete gynecological history and a gynecological
physical examination were conducted at day 1, and at Months
7, 12, 24, and 36 [20]. Genital specimens were obtained from
all subjects at day 1 and at Months 7, 12, 18, 24, 30, and 36
for HPV DNA detection as described [20].
An interim analysis was planned for the time when approximately 50% of the postdose 3 (Month 7) responses from Part
B were available. The purpose of this interim analysis was to
select a dose of quadrivalent vaccine for Phase III studies.
2.3. HPV Serologic assays
Serum antibodies to HPV 6, 11, 16 and 18 were measured
using a competitive radioimmunoassay (cRIA) or a competitive Luminex immunoassay (cLIA) in both vaccine and
placebo groups [23–25]. Serum samples were obtained from
all subjects at day 1 and at Months 2, 3, 6, 7, 12, 18, 24, 30,
and 36. Under a Protocol Amendment, the assay method was
changed from cRIA to cLIA. The optimization and validation of the assay and cRIA-to-cLIA concordance is published
elsewhere [23,25]. Prior to the Protocol Amendment, all day
1 and Months 2, 3, 6 and 7 samples were tested by cRIA. After
the Protocol Amendment, the cLIA assay was used consistently for the analysis of only one dose formulation of vaccine
(i.e., 20/40/40/20 ␮g dose) over 36 months of follow-up and
included testing of samples from day 1 and Months 2, 3, 6,
7, 12, 18, 24, 30, and 36.
2.4. HPV competitive radioimmunoassays (cRIAs)
Four separate HPV cRIAs (one assay per HPV type),
developed by Merck Research Laboratories, were used to
quantitate serum HPV 6, 11, 16 and 18 specific antibodies as
described [24]. Briefly, polystyrene beads were coated with
a limiting amount of HPV 6, 11, 16 or 18 L1 VLP antigen, incubated with the corresponding HPV 6, 11, 16 or 18
specific mouse MAbs (H6.B10.5 [26], H11.B2 [27], H16.V5
[28] and H18.J4 [28], respectively) and a serum sample. Goat
anti-mouse antibodies tagged with 125 I were used to detect the
amount of mouse MAb bound to the VLPs. Standard curves
were constructed using dilutions of serum from an HPV 6,
11 or 18 L1 VLP immunized African Green Monkey or an
HPV 16 L1 VLP immunized Chimpanzee. As a competitive
assay, the measure of VLP bound MAb is an indirect measurement of the presence of serum antibody to a neutralizing
epitope of the VLP. Sample titers were interpolated from the
four-parameter logistic regression fit to the standard curve,
corrected for dilution, and reported in arbitrary units (milliMerck Units per milliliter [mMU/mL]). The lower limits of
quantitation were 4.0, 6.5, 1.3, and 6.5 mMU/mL and the
serostatus cutoffs were 8.0, 13.0, 6.0 and 13.0 mMU/mL for
antibodies to HPV 6, 11, 16 and 18, respectively.
2.5. HPV competitive luminex immunoassay (HPV cLIA)
The HPV cLIA is a competitive assay that measures in a
single serum sample HPV 6, 11, 16 and 18 type-specific anti-
bodies to conformationally sensitive, neutralizing epitopes on
HPV L1 VLPs [25]. The assay uses yeast-derived HPV L1
6, 11, 16 and 18 VLPs that have been covalently conjugated
to Luminex microspheres 6, 11, 16 and 18, respectively. The
type-specific HPV-VLP antibody responses are associated
with specific Luminex microspheres that are identified by
their distinct red and infrared fluorescent dye spectral properties on a Luminex100 BioPlex instrument. Antibody titers
are determined in a competitive format, where known, typespecific phycoerythrin (PE)-labeled, neutralizing antibodies
H6.M48 [26] for HPV 6, K11.B2 [25] for HPV 11, H16.V5
[28] for HPV 16 and H18.J4 [28] for HPV 18, compete
with patient serum antibodies for binding to conformationally
sensitive, neutralizing epitopes on the VLPs. For each HPV
type, relative inhibition of mAb-PE binding is compared to
a pooled standard reference serum using a four-parameter
logistic curve fit [29], corrected for dilution and reported in
mMU/mL. The lower limits of quantitation were 8.0, 8.0, 12.0
and 8.0 mMU/mL and the serostatus cutoffs were 20, 16, 20
and 24 mMU/mL for HPV 6, 11, 16 and 18, respectively.
2.6. Statistical analysis
The immunogenicity objectives of this report were: (1)
to report the results of the pre-specified interim summary of
immune responses for dose selection of quadrivalent vaccine
for use in Phase III studies; (2) to report the results of an
exploratory evaluation of immune responses over time for the
dose selected for Phase III development; and (3) to report the
results of an exploratory comparison of immune responses
between subjects that were naı̈ve to a given vaccine HPV
type at day 1 and subjects with serologic evidence of having
cleared a prior infection with the same vaccine HPV type. All
of these evaluations were exploratory in nature and therefore
no statistical tests were performed.
When approximately 50% of the postdose 3 (Month 7)
responses as measured by cRIA were available from subjects enrolled in Part B (June 2001), an interim summary of
immunogenicity and safety was conducted in subjects who
were seronegative (by cRIA) and PCR negative to the relative
vaccine HPV type at day 1. Subjects in the placebo arms were
pooled for this analysis. For the interim summary of immunogenicity, anti-HPV levels in seronegative subjects were set to
4.0, 6.5, 3.0 and 6.5 mMU/mL, respectively. For consistency
of analysis, the cRIA assay was used for the interim summary
through Month 7.
Geometric mean titers (GMTs) and their associated 95%
confidence intervals, and longitudinal plots were used in evaluating the immunogenicity of the quadrivalent HPV (Types
6/11/16/18) L1 VLP vaccine 20/40/40/20 ␮g dose. For consistency of analysis, the cLIA assay was used for the analysis
of this one, low-dose of vaccine over the entire 36 months
of follow-up. GMTs were measured in three cohorts: (1)
the per-protocol immunogenicity (PPI) cohort which consisted of subjects who were naı̈ve to the relevant HPV type at
enrollment, remained free of infection with the same vaccine
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
HPV type through the completion of the vaccination regimen, received all 3 doses of vaccine or placebo within the
protocol specified time frames (vaccination 2 = 44–76 days
relative to vaccination 1; vaccination 3 = 145–215 days relative to vaccination 1; Month 7 serum sample collected within
14–46 days following the third vaccination) and did not violate the protocol; (2) a cohort consisting of subjects who
were seropositive, but PCR negative to the relevant HPV
type at enrollment; and (3) a cohort of subjects who were
seronegative, but PCR positive to the relevant HPV type
at enrollment. For the PPI population, percentages of subjects who seroconverted at Month 7 and subsequently were
seropositive at Months 18 and 36 were calculated. In addition, the percentages of subjects who remained seropositive
at Months 18 and 36 were determined among quadrivalent
vaccine recipients who were seropositive and PCR negative
to the relevant vaccine HPV type at enrollment, who received
all three vaccinations, and had a Month 7 serum sample collected. In addition, the Month 7 antibody responses in the
PPI population were compared by region (USA, Brazil, and
Europe).
With the exception of a designated unblinded statistician,
in-house blinding was maintained until the completion of the
study. The designated unblinded statistician was appointed
to perform the safety and immunogenicity interim analysis
on data through the Month 7 time point. Subjects, investigators (and their staff), HPV vaccine clinical, data management,
statistics, regulatory, and quality assurance personnel at the
Sponsor, Pathology Panel members, and laboratory personnel
conducting the PCR and serology assays were not informed
of individual vaccination allocations until the end of the study.
Dose selection for future studies was not disclosed to investigators until the initiation of the Phase III program (December
2001).
The primary safety hypothesis for Part B of the study stated
that quadrivalent HPV L1 VLP vaccine is generally welltolerated. If no serious vaccine-related adverse experiences
were observed among the 250 subjects in each quadrivalent
vaccine dose group, there was a 97.5% probability that the
true rate is <1.5%. The proportion of subjects with serious
adverse experiences and all adverse experiences were summarized by vaccination group and type of adverse experience.
Per-protocol, p-values were computed for those prompted
adverse experiences on the VRC, including injection-site
pain, erythema and redness. Due to differing concentrations of AAHS in the various vaccine and placebo treatment
groups, subjects who received the low- and intermediatedoses of quadrivalent vaccine (containing 225 ␮g AAHS)
were compared to the low-dose placebo formulation (225 ␮g
AAHS), while subjects who received the high-dose formulation of vaccine (containing 395 ␮g AAHS) were compared
to the high-dose placebo formulation (450 ␮g AAHS). The
method of Miettinen and Nurminen [30] was used for all comparisons of safety profiles among the vaccination groups.
The safety profiles were also summarized in two subpopulations: (1) subjects who were seronegative at day 1 and
5575
PCR negative from day 1 through Month 7 to all vaccine
HPV types; and (2) subjects who were seropositive at day
1 or PCR positive to any of the vaccine HPV types at any
time from day 1 through Month 7, to evaluate the impact of
baseline HPV 6, 11, 16 or 18 status on the tolerability of the
quadrivalent HPV vaccine.
3. Results
3.1. Baseline demographics
The baseline demographic characteristics were generally
well-balanced among the five vaccination groups (Table 1).
These 13–24 year-old women were predominantly Caucasians and approximately 25% (277/1106) were current
smokers. The mean age at first sexual intercourse was
between 16 and 17 for each vaccination group. Over 60%
(707/1106) of subjects reported fewer than 3 lifetime sexual
partners at enrollment. The percentages of subjects who used
barrier, behavior, hormonal, and other contraceptive methods
were comparable among the five vaccination groups. The
most common contraceptive methods were hormonal contraceptives (640/1106; 57.9%) and male condom (271/1106;
24.5%).
The percentages of subjects who were seropositive or PCR
positive to at least one of the vaccine HPV types at baseline
were generally comparable among the vaccination groups
(Table 2). For the overall study cohort, approximately 18.2%
(201/1103) and 12.1% (133/1100) of these women were antiHPV 6, 11, 16 or 18 seropositive and HPV 6, 11, 16 or 18 DNA
positive at baseline, respectively. Similarly, within each HPV
type, seropositivity was more prevalent than PCR positivity.
At baseline, more subjects were seropositive or PCR positive
to HPV 16 than to the other three vaccine HPV types. In
general, the percentages of subjects that had abnormal Pap
test diagnoses (8.9% to 12.1%) were comparable among the
five vaccination groups (Table 1).
3.2. Primary Dose Selection
The primary immunogenicity objective was to select
one of three formulations of a quadrivalent HPV (Types
6/11/16/18) L1 VLP vaccine for use in Phase III studies
[20]. Based on the interim summary of immune responses,
for each of the four components, the postdose 3 (Month 7)
anti-HPV GMTs were ∼27- to 145-fold higher than those
observed in placebo recipients who were vaccine-type HPV
seropositive at day 1 (Fig. 2). For each of the four components, anti-HPV GMTs at Month 7 appeared to be comparable among the three formulations of quadrivalent vaccine
(Fig. 2). For all three formulations, 100% of subjects seroconverted (i.e., showed evidence of vaccine-induced immune
responses) by Month 7. The formulation comprising the lowest dose (20/40/40/20 ␮g) of HPV 6, 11, 16 and 18 L1 VLPs
was therefore chosen for evaluation in Phase III studies.
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L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
Table 1
Summary of subject characteristics by vaccination group at enrollment
Age (years)
Mean ± S.D.
Range
Ethnicity
Asian
Black
Hispanic American
Native American
White
Other
Placebo (aluminum adjuvant)
Quadrivalent HPV (Types 6/11/16/18) L1 VLP vaccine
Placebo (225 ␮g),
N = 135, n (%)a
20/40/40/20 ␮g,
N = 277, n (%)a
19.9 ± 1.7
16–23
Placebo (450 ␮g),
N = 140, n (%)a
20.0 ± 1.8
13–23
20.2 ± 1.7
16–23
40/40/40/40 ␮g,
N = 274, n (%)a
20.0 ± 1.7
15–24
80/80/40/80 ␮g,
N = 280, n (%)a
20.0 ± 1.8
16–23
Total N = 1106,
n (%)a
20.0 ± 1.7
13–24
6 (4.4)
11 (8.1)
10 (7.4)
0 (0.0)
101 (74.8)
7 (5.2)
5 (3.6)
7 (5.0)
10 (7.1)
1 (0.7)
113 (80.7)
4 (2.9)
7 (2.5)
25 (9.0)
14 (5.1)
2 (0.7)
216 (78.0)
13 (4.7)
11 (4.0)
32 (11.7)
14 (5.1)
4 (1.5)
204 (74.5)
9 (3.3)
4 (1.4)
26 (9.3)
10 (3.6)
2 (0.7)
230 (82.1)
8 (2.9)
33 (3.0)
101 (9.1)
58 (5.2)
9 (0.8)
864 (78.1)
41 (3.7)
Smoking status
Never smoked
Ex-smoker
Current smoker
Unknown
92 (68.1)
15 (11.1)
27 (20.0)
1 (0.7)
79 (56.4)
18 (12.9)
42 (30.0)
1 (0.7)
183 (66.1)
29 (10.5)
64 (23.1)
1 (0.4)
169 (61.7)
31 (11.3)
72 (26.3)
2 (0.7)
170 (60.7)
38 (13.6)
72 (25.7)
0 (0)
693 (62.7)
131 (11.8)
277 (25.0)
5 (0.5)
National origin
USA
Brazil
Europe
63 (46.7)
46 (34.0)
26 (19.3)
63 (45.0)
47 (33.6)
30 (21.4)
125 (45.1)
94 (33.9)
58 (20.9)
123 (44.9)
91 (33.2)
60 (21.9)
127 (45.4)
94 (33.6)
59 (21.1)
501 (45.3)
372 (33.6)
233 (21.1)
Age at first sexual intercourse (years)
Mean ± S.D.
16.6 ± 1.8
Median (range)
17 (13–23)
Lifetime number of sexual partners at enrollment
0
6 (4.4)
1
47 (34.8)
2
41 (30.4)
3
19 (14.1)
4
22 (16.3)
Median (range)
2 (0–4)
Pap test
Without Pap test at day 1
Unsatisfactory
Normal
Abnormal
ASCUS
LSIL
HSIL
AGC
Contraception methodb
Male condom
Behavioral
Hormonal
Other
16.7 ± 1.8
17 (12–23)
16.7 ± 1.8
16 (12–22)
10 (7.1)
41 (29.3)
34 (24.3)
31 (22.1)
24 (17.1)
2 (0–4)
17 (6.1)
80 (28.9)
73 (26.4)
67 (24.2)
40 (14.4)
2 (0–4)
16.8 ± 1.9
17 (13–23)
25 (9.1)
74 (27.0)
83 (30.3)
51 (18.6)
41 (15.0)
2 (0–4)
16.8 ± 1.9
17 (11–22)
18 (6.4)
88 (31.4)
70 (25.0)
62 (22.1)
41 (14.6)
2 (0–4)
16.7 ± 1.8
17 (11–23)
76 (6.9)
330 (29.8)
301 (27.2)
230 (20.8)
168 (15.2)
2 (0–4)
3 (2.2)
1 (0.7)
119 (88.1)
12 (8.9)
6 (4.4)
5 (3.7)
1 (0.7)
0 (0.0)
3 (2.1)
1 (0.7)
119 (85.0)
17 (12.1)
11 (7.9)
5 (3.6)
1 (0.7)
0 (0.0)
10 (3.6)
3 (1.1)
231 (83.4)
33 (11.9)
16 (5.8)
15 (5.4)
1 (0.4)
1 (0.4)
10 (3.6)
4 (1.5)
229 (83.6)
31 (11.3)
11 (4.0)
18 (6.6)
2 (0.7)
0 (0.0)
8 (2.9)
3 (1.1)
239 (85.4)
30 (10.7)
14 (5.0)
14 (5.0)
2 (0.7)
0 (0.0)
34 (3.1)
12 (1.1)
937 (84.7)
123 (11.1)
58 (5.2)
57 (5.2)
7 (0.6)
1 (0.1)
40 (29.6)
23 (17.0)
76 (56.3)
8 (5.9)
36 (25.7)
25 (17.9)
81 (57.9)
9 (6.4)
63 (22.8)
48 (17.4)
161 (58.3)
21 (7.6)
58 (21.3)
51 (18.8)
163 (59.9)
14 (5.1)
74 (26.4)
50 (17.9)
159 (56.8)
17 (6.1)
271 (24.5)
1497 (17.8)
640 (57.9)
69 (6.2)
SIL, squamous intraepithelial lesion; ASCUS, atypical squamous cells of undetermined significance; LSIL, low-grade SIL; HSIL, high-grade SIL; AGC,
atypical glandular cells; N, number of subjects randomized to the respective vaccination group; n, number of subjects with the indicated characteristic.
a Percentage was computed as 100 × (n/N).
b The same subject may appear in more than one category.
3.3. Immunogenicity of the Phase III formulation
Of the 552 participants randomized to the Phase III formulation and pooled placebo arms, 551 received at least one
injection of vaccine or placebo. Among these subjects, 406,
379, and 428 were included in the PPI analyses for Types
6/11, 16, and 18, respectively [20]. The primary reason for
exclusion from the PPI analysis was detection of vaccinetype HPV antibodies at day 1 and/or detection of vaccine-type
HPV DNA at day 1 through Month 7, followed by general
protocol violations (57 subjects). The most common protocol
violation was an incomplete vaccination series (30 subjects)
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
5577
Table 2
Summary of anti-HPV serostatus and HPV PCR status at day 1 by vaccination group and HPV type
Pooled placebo (250 ␮g + 450 ␮g),
N = 275, m/n (%)
20/40/40/20 ␮g,
N = 277, m/n (%)
40/40/40/40 ␮g,
N = 274, m/n (%)
80/80/40/80 ␮g,
N = 280, m/n (%)
Total N = 1106,
m/n (%)
21/275 (7.6)
5/275 (1.8)
16/276 (5.8)
8/275 (2.9)
21/272 (7.7)
14/271 (5.2)
15/280 (5.4)
3/279 (1.1)
73/1103 (6.6)
30/1100 (2.7)
6/275 (2.2)
3/275 (1.1)
6/276 (2.2)
2/275 (0.7)
8/272 (2.9)
5/271 (1.8)
5/280 (1.8)
2/279 (0.7)
25/1103 (2.3)
12/1100 (1.1)
29/275 (10.5)
25/275 (9.1)
32/276 (11.6)
24/275 (8.7)
31/272 (11.4)
24/271 (8.9)
24/280 (8.6)
17/279 (6.1)
116/1103 (10.5)
90/1100 (8.2)
HPV 18
Seropositive
DNA positive
12/275 (4.4)
4/275 (1.5)
10/276 (3.6)
4/275 (1.5)
12/272 (4.4)
6/271 (2.2)
12/280 (4.3)
6/279 (2.2)
46/1103 (4.2)
20/1100 (1.8)
HPV 6, 11, 16 or 18
Seropositive
DNA positive
51/275 (18.5)
32/275 (11.6)
50/276 (18.1)
32/275 (11.6)
57/272 (21.0)
43/271 (15.9)
43/280 (15.4)
26/279 (9.3)
201/1103 (18.2)
133/1100 (12.1)
Day 1 status
HPV 6
Seropositive
DNA positive
HPV 11
Seropositive
DNA positive
HPV 16
Seropositive
DNA positive
N, number of subjects randomized to the respective vaccination group; m, number of subjects contributing to the analysis; n, number of subjects with a valid
or non-missing day 1 immunoassay or PCR result.
Fig. 2. Interim analysis of immunogenicity of three formulations of quadrivalent vaccine. Postdose 3 (Month 7) anti-HPV cRIA GMTs (mMU/mL with 95%
confidence intervals on a logarithmic scale) are shown for: (a) HPV 6; (b) HPV 11; (c) HPV 16; and (d) HPV 18. Because the titers of the reference sera are
not identical, one cannot draw conclusions from the absolute titers with regard to the relative immunogenicity of the four VLP components in the vaccine; n is
the number of subjects contributing to the analysis.
5578
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
Fig. 3. Anti-HPV cLIA GMTs (mMU/mL with 95% confidence intervals) for quadrivalent vaccine (Phase III formulation) and placebo recipients (pooled
placebo arms). Longitudinal plots are shown by HPV type for the PPI population and for subjects who were baseline seropositive and PCR negative: (A) HPV
6; (B) HPV 11; (C) HPV 16; and (D) HPV 18. Because the titers of the reference sera are not identical, one cannot draw conclusions from the absolute titers
with regard to the relative immunogenicity of the four VLP components in the vaccine. n = number of subjects contributing to the Month 36 time point.
or vaccination 2 or 3 out of the protocol-specified time window (26 subjects).
Baseline vaccine-type HPV naı̈ve subjects who received
the 20/40/40/20 ␮g formulation of quadrivalent vaccine
mounted a robust immune response (Fig. 3, Table 3). For comparison, antibody titers for baseline seropositive and DNA
negative subjects who received placebo are also plotted in
Fig. 3. This population is enriched for subjects who had
presumably been infected with the relevant vaccine HPV
type, mounted an immune response to the infection, and
then cleared that infection prior to enrollment. The GMTs
observed in these subjects provide a reference against which
to evaluate vaccine-induced anti-HPV responses. Although
the total number of women in these subpopulations was small,
the HPV 6, 11, 16 and 18 antibody titers in these baseline
seropositive and PCR negative subjects who received placebo
remained constant throughout the 3-year period of the
study.
One month after the primary dose (Month 2) vaccineinduced antibody levels were above those seen in the baseline
seropositive and DNA negative placebo subjects for the HPV
16 component only. However, upon completion of a 3-dose
regimen, anti-HPV 6, 11, 16 and 18 antibody levels were
∼10- to 104-fold higher than those observed in these placebo
recipients who were vaccine-type HPV seropositive at enrollment. Furthermore, 100% of vaccine recipients with a valid
Month 7 serum result had detectable HPV 6, 11, 16 and 18
antibodies. As shown in Fig. 3, the vaccine-induced immune
response appears highest for HPV 16, however direct comparisons of the relative immunogenicity of the four VLP
components cannot be made from the absolute titers, as the
titers for each of the reference sera for the individual assays
were not identical.
Following an initial, similar sized decline, vaccineinduced HPV 6, 11, 16 and 18 antibody levels plateaued
and remained stable for at least 2.5 years postdose 3 for all
four types. In the PPI population, anti-HPV 6, 11, 16 or 18
GMTs at Month 24 (93.7, 99.8, 420.6 and 59.9 mMU/mL,
respectively) were comparable to those observed at Month
36 (93.4, 94.0, 508.8 and 59.7 mMU/mL, respectively). At
Month 36, vaccine-induced antibody titers reached that of
natural infection for HPV 6, 11, and 18, while HPV 16 antibody titers remained ∼17-fold higher than that of natural
infection (Fig. 3C). As shown in Fig. 3, average serum HPV
antibody levels remained below the limits of quantitation in
baseline vaccine-type HPV-naı̈ve placebo recipients.
Administration of quadrivalent vaccine to women with
detectable HPV antibodies at enrollment resulted in higher
anti-HPV responses than those observed among quadrivalent vaccine recipients who were naı̈ve to the relevant HPV
type at baseline, although the 95% confidence intervals overlapped at most time points (Fig. 3, Table 3). The difference
was particularly evident after the first vaccine injection: At
Month 2, among women with detectable anti-HPV 6, 11,
16 or 18 antibodies at baseline, vaccine-induced anti-HPV
responses were ∼12- to 26-fold higher than those observed
in the baseline-naı̈ve vaccine recipients (i.e., the PPI population). Taken together, these results suggest that women
who were baseline anti-HPV positive had developed a booster
response to the vaccination.
To further investigate the long-term immunogenicity of
the Phase III formulation, percentages of per-protocol subjects who seroconverted at Month 7 and subsequently were
seropositive at Months 18 and 36 were calculated. At Month
7, for each vaccine HPV type, 100% of subjects who received
the vaccine in the PPI population became seropositive for
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
5579
Table 3
Summary of anti-HPV cLIA titers among the PPI population, and subjects who were HPV seropositive/PCR negative to the corresponding HPV vaccine-type
at day 1
Cohort
n
Day 1 serology status
Day 1 DNA status
HPV 6
Month 2
Month 2
Month 7
Month 7
Month 36
Month 36
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
HPV 11
Month 2
Month 2
Month 7
Month 7
Month 36
Month 36
Negative
Positive
Negative
Positive
Negative
Positive
HPV 16
Month 2
Month 2
Month 7
Month 7
Month 36
Month 36
HPV 18
Month 2
Month 2
Month 7
Month 7
Month 36
Month 36
Phase III formulation (N = 276)
GMT (mMU/mL)
95% CI
208
14
208
13
184
13
32
821
582
1102
93
237
(27–37)
(233–2886)
(527–643)
(504–2410)
(81–108)
(97–582)
Negative
Negative
Negative
Negative
Negative
Negative
208
6
208
6
184
6
37
477
697
1364
94
375
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
194
20
194
17
177
14
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
219
10
219
10
196
9
n
Pooled placebo arms (N = 275)
GMT (mMU/mL)
95% CI
197
18
198
17
184
16
4.6
56
4.6
55
5.1
68
(4.3–4.9)
(32–99)
(4.3–4.8)
(28–108)
(4.7–5.6)
(33–139)
(32–43)
(40–5707)
(618–785)
(550–3383)
(81–110)
(113–1248)
197
4
198
4
184
4
4.1
95
4.1
94
4.4
96
(4.0–4.3)
(2.9–3145)
(4.0–4.2)
(5.4–1639)
(4.1–4.7)
(19–498)
147
1865
3892
4575
509
985
(123–177)
(687–5061)
(3324–4558)
(2573–8135)
(436–593)
(524–1854)
184
16
185
15
170
15
6.4
27
6.5
37
7.7
29
(6.0–6.9)
(12–63)
(6.2–6.9)
(17–85)
(6.8–8.8)
(12–69)
14
268
801
687
60
94
(12–16)
(68–1052)
(694–925)
(413–1142)
(49–74)
(41–214)
208
12
209
12
193
10
4.5
42
4.6
42
4.8
29
(4.2–4.9)
(22–79)
(4.3–5.0)
(23–75)
(4.4–5.2)
(15–59)
N, number of subjects randomized to the respective vaccination group who received at least one injection; n, number of subjects contributing to the analysis.
the relevant vaccine HPV type. Among subjects who had
valid immunoassay results, 98%, 98% 100% and 86% were
seropositive for HPV Types 6, 11, 16 and 18 at Month
18; and 94%, 96%, 100% and 76% were seropositive for
HPV Types 6, 11, 16 and 18 at Month 36, respectively. In
comparison to the PPI population, among quadrivalent vaccine recipients who were seropositive and PCR negative to
the relevant vaccine HPV type at enrollment 85% (11/13),
100% (6/6), 100% (15/15), and 100% (9/9) were seropositive for HPV Types 6, 11, 16 and 18 at Month 18; and
92% (12/13), 100% (6/6), 100% (13/13) and 88% (8/9) were
seropositive for HPV Types 6, 11, 16 and 18 at Month 36,
respectively.
The anti-HPV serum cLIA responses among subjects who
were baseline PCR positive and seronegative to the relevant HPV type(s) were also measured. These subjects had
vaccine-type HPV DNA detected at day 1 but did not have
measurable antibodies to the same type. Anti-HPV 6, 11, 16,
and 18 GMTs in these subjects appeared to be comparable to
those observed in the PPI population (data not shown). However, the small sample size in this study limited the capacity
to detect potential differences in vaccine-induced immune
responses between the groups.
The Month 7 antibody responses in the PPI population
were compared by geographic region. There was no apparent
difference in vaccine immunogenicity among women from
the United States, Europe, and Brazil (data not shown).
3.4. Safety
Safety data for the low-dose Phase III formulation has
been described [20]. A detailed comparison of safety data for
all three formulation of quadrivalent vaccine is provided in
Table 4. As shown in Table 4, the proportion of subjects who
reported any adverse experience (day 1 through day 15 following any vaccination visit) was slightly increased among
the quadrivalent HPV vaccine groups compared with the
placebo groups. The incidences of systemic clinical adverse
experiences were generally comparable among the five vaccination groups. The most commonly reported systemic clinical adverse experiences judged by the site investigator to be
vaccine-related were headache and pyrexia. The most commonly reported injection-site adverse experiences across the
5 vaccination groups were injection-site pain, erythema, and
swelling. A total of six serious adverse experiences were
reported. None was judged to be vaccine-related. Within
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L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
Table 4
Clinical adverse experience summary (days 1–15 following any vaccination visit)
Subjects with follow-up
With one or more AE
With one or more systemic AE
Gastrointestinal disorders
Fatigue
Dizziness
Headache
Rash
Pyrexia
With one or more injection-site AE
Injection site bruising
Injection site erythema
Injection site painb
Injection site pruritus
Injection site reaction
Injection site swellingc
With serious AE
With serious vaccine-related AE
Placebo (aluminum adjuvant)
Quadrivalent HPV (Types 6, 11, 16, 18) L1 VLP vaccine
Placebo (225 ␮g),
N = 135, n (%)a
Placebo (450 ␮g),
N = 140, n (%)a
20/40/40/20 ␮g,
N = 275, n (%)a
40/40/40/40 ␮g,
N = 272, n (%)a
80/80/40/80 ␮g,
N = 280, n (%)a
134
116 (86.6)
140
126 (90.0)
272
250 (91.9)
269
251 (93.3)
277
265 (95.7)
95 (70.9)
29 (21.6)
9 (6.7)
1 (0.7)
48 (35.8)
1 (0.7)
13 (9.7)
95 (67.9)
30 (21.4)
8 (5.7)
5 (3.6)
54 (38.6)
2 (1.4)
16 (11.4)
187 (68.8)
67 (24.6)
15 (5.5)
13 (4.8)
110 (40.4)
3 (1.1)
30 (11.0)
186 (69.1)
69 (25.7)
8 (3.0)
9 (3.3)
98 (36.4)
5 (1.9)
37 (13.8)
192 (69.3)
66 (23.8)
11 (4.0)
9 (3.2)
112 (40.4)
0 (0.0)
41 (14.8)
100 (74.6)
6 (4.5)
26 (19.4)
98 (73.1)
1 (0.7)
0 (0.0)
20 (14.9)
0 (0.0)
0 (0.0)
112 (80.0)
3 (2.1)
30 (21.4)
111 (79.3)
3 (2.1)
0 (0.0)
29 (20.7)
2 (1.4)
0 (0.0)
234 (86.0)
13 (4.8)
73 (26.8)
232 (85.3)
8 (2.9)
0 (0.0)
76 (27.9)
2 (0.7)
0 (0.0)
240 (89.2)
7 (2.6)
58 (21.6)
238 (88.5)
11 (4.1)
0 (0.0)
72 (26.8)
0 (0.0)
0 (0.0))
255 (92.1)
9 (3.2)
75 (27.1)
251 (90.6)
9 (3.2)
5 (1.8)
89 (32.1)
2 (0.7)
0 (0.0)
N, number of subjects randomized to the respective vaccination group who received at least one injection; n, number of subjects within category.
a Percentage was computed as 100 × (n/number of subjects with follow-up).
b p = 0.003, <0.001, and 0.001 for the low-, intermediate-, and high-dose, respectively.
c p = 0.004, 0.008, and 0.015 for the low-, intermediate-, and high-dose, respectively.
each vaccination group, the majority (approximately 95%,
5430/5797) of the reported adverse experiences were mild or
moderate in intensity.
When compared with the corresponding placebo group,
the percentages of subjects who had injection-site pain and
injection-site swelling were statistically higher among the
recipients of active quadrivalent vaccine (Table 4). It should
be noted that no multiplicity adjustment was made to control
the overall type I error rate for these comparisons.
Among the active vaccine recipients, the rates of clinical adverse experiences, injection-site adverse experiences,
or systemic adverse experiences tended to be lower among
subjects who were seropositive at day 1 or PCR positive from
day 1 through Month 7 to any vaccine HPV type, as compared
to subjects who were seronegative at day 1 and PCR negative
from day 1 through Month 7 to all vaccine HPV types (data
not shown).
4. Discussion
The duration of protection afforded by vaccines represents
a critical test of their utility as public health interventions.
Some vaccines induce long-term immunity. Others require
the administration of booster doses. Experience with hepatitis
B vaccine may provide some guidance on long-term immunity raised by HPV vaccination since these two vaccines are
similar in design. Vaccine-induced antibody against hepatitis
B wanes over the course of several years, but re-exposure to
antigen results in a rapid anamnestic response, or memory
immune response [31]. In field studies, good long-term protection in absence of any booster has been seen against both
infection and, moreover, chronic carriage [32,33].
In the current study we included a cohort of women that
was sexually active, at risk for acquisition of HPV 6, 11,
16, and 18 infections and was broadly comparable to the
general population of young women in developed and developing countries [34,35]. Unlike other vaccine studies [19] we
had the opportunity to examine immune responses among
HPV seropositive women at baseline, as women were not
prescreened for HPV DNA or seropositivity prior to enrollment. In this study, as well as in a previous study focusing on
a monovalent HPV 16 L1 VLP vaccine [21], women with
detectable vaccine-type anti-HPV levels prior to vaccination were shown to respond with faster rises, higher peaks,
and higher persistence levels following administration of
HPV vaccine compared to baseline HPV naı̈ve women. The
results for the monovalent HPV 16 vaccine, in addition to
the results presented here for a quadrivalent vaccine, suggest
that HPV L1 VLP vaccines formulated on aluminum adjuvant can induce anamnestic responses. Such responses may
allow for long-term vaccine-induced protection, either following a primary series, or with booster vaccinations. To our
knowledge this is the first report demonstrating that administration of a quadrivalent HPV (6/11/16/18) L1 VLP vaccine to
women with detectable HPV antibodies prior to vaccination
results in an anamnestic response. In addition, the vaccine
was generally well-tolerated in women who were infected
with vaccine-HPV types prior to vaccination. However, a
limitation of the present study is the small number of sub-
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
jects who were seropositive at day 1 or PCR positive to any
of the vaccine HPV types. Therefore, these data should be
considered preliminary.
The serology assays used in these studies measured HPV
antibody titers in a competitive format whereby the serum
antibodies compete with HPV type-specific mouse MAbs to
neutralizing epitopes present on each VLP. In the dose selection phase of the study, serum antibodies to HPV 6, 11, 16
and 18 were measured using a competitive radioimmunoassay. Following dose selection, under a Protocol Amendment,
the assay method was changed from the cRIA to a competitive Luminex immunoassay. The switch from the cRIAs to
the cLIAs following dose selection occurred for several reasons. First and foremost, the radio-immunoassay kits were
being discontinued by the manufacturer. Second, the cRIAs
used radioactive 125 I-labeled secondary antibodies whereas
fluorescent-based technologies represented a safer alternative. Third, the cRIAs were labor intensive whereas the
Luminex assay was amenable to high-throughput screening. Lastly, the microsphere-based fluorescence technology is
considerably more precise than the historical RIAs. Although
the serostatus cutoffs are slightly different between the cRIA
and cLIA assays, due to differences in the platform technologies, VLPs and MAbs used in the individual assays, these
differences do not impact the findings of this study, as the
cRIA data was used consistently for dose selection whereas
the cLIA was used consistently for comparing long-term
immunogenicity data for the low-dose phase III formulation.
Regardless of the type of assay used (cLIA or cRIA), the
antibody titers of the four HPV types in the reference sera
are not identical between the cRIA and cLIA. In addition,
the four mAbs used in the four assays recognize unique epitopes, therefore one can not draw conclusions with regard
to the relative immunogenicity of the four VLP components in the vaccine. In addition, antibody titer levels should
not be compared across vaccine studies in which different
HPV antibody assays are conducted [19]. Antibodies are
generated against a number of epitopes on the VLPs. The
mAbs used in the competitive assays each recognize typespecific epitopes. Overall, some epitopes appear to be more
immunogenic than others, however the immunogencity of
entire VLPs appears to be about the same. The four mAbs
used in the four-plex cLIA assay each recognize epitopes
having a unique binding affinity and a unique ranking in the
total immunogloblulin immunodominance spectra for their
respective HPV VLP type. Therefore, the scale of the competitive immune response is dependent upon the particular
attributes of the mAbs and the epitope that they recognize.
It is fairly uncommon that a vaccine will produce an
immune response greater than that achieved by natural infection. The fact that a persistent and measurable immune
response equal to or greater than that observed during natural infection is encouraging with regard to the utility and
longevity of the vaccine response. Again, the scale of persistent titers achieved for the four types should not be compared with one another. The immunodominance of the epi-
5581
tope being monitored and the mAb binding affinities are
independent type-specific factors affecting the scale of the
titer noted. Vaccine studies in rhesus macaques suggest
that the four VLPs are equally immunogenic and that the
differences in titers measured by cRIA or cLIA are due
to differences in the assigned potencies of the reference
sera [36]. The fact that the relative changes in the GMTs
for all the vaccine HPV types are remarkably similar over
time suggests that the immunogenicity of the four HPV
VLPs is probably comparable. A measurement of the total
IgG response demonstrates immunogenicity, but does not
assess the utility of the antibodies generated. The advantage of using a competitive assay, rather than measuring
the total IgG response to vaccine VLPs by any variety of
assays such as ELISA or Luminex, is that it allows one to
monitor the antibody response to known neutralizing epitopes [25,26], thus providing a specific measure of immunogenicity that is clinically related to protection against HPV
infection.
In developed countries, the greatest risk for acquisition of
HPV infections is correlated to the period subsequent to sexual debut. Women remain at risk for HPV infection as long
as they continue to have genital skin contact with other HPV
infected skin. Based on the immunogenicity and efficacy
findings [20] of this study, it is clear that administration of
quadrivalent HPV (Types 6/11/16/18) L1 VLP vaccine confers protective efficacy for at least 2.5 years post-vaccination.
Among placebo recipients who were anti-HPV 6, 11, 16 or
18 seropositive, antibody levels remained stable during this
3-year study. In the PPI-population there was a decline in
seropositivity over time. The decline in seropositivey was
highest for HPV 18, however there were no breakthrough
cases of HPV 18-related cervical intraepithelial neoplasia in
quadrivalent vaccine recipients [20]. The stability of both
vaccine-induced and infection-induced anti-HPV levels over
time suggests that vaccine-induced protective efficacy will be
long-lasting. Long-term follow-up of HPV vaccine studies
will provide information on the duration of efficacy. Ongoing phase III efficacy studies have the potential to detect
breakthrough HPV disease in a large population of vaccinated women and to confirm the long-term duration of the
immune response and the potential correlation between antiHPV levels and vaccine efficacy.
Results from this dose-ranging, quadrivalent HPV
6/11/16/18 vaccine trial showed that all three vaccine doses
were highly immunogenic and generally safe and welltolerated. Thus, the lowest dose was selected for licensure
evaluation in Phase III clinical trials. This dose of vaccine
also appeared to stimulate an anamnestic response among
the subcohorts of immunized women who had serological
evidence of prior infection with a vaccine HPV type at baseline. Worldwide Phase III studies of this quadrivalent vaccine
(GARDASIL® ) in more than 25,000 subjects are underway.
These studies will provide a definitive evaluation of the vaccine’s impact on HPV-related clinical disease in adolescents
and in young adult women. A primary focus on this age group
5582
L.L. Villa et al. / Vaccine 24 (2006) 5571–5583
for vaccination is supported by a peak of infection in the early
twenties, expected higher effectiveness of the vaccine prior
to exposure and easier access to public health interventions in
early teens rather than in adult age. However, including older
women and young men in a universal immunization policy
against cervical cancer and genital warts, as a complement to
cervical screening, also warrants investigation.
Acknowledgements
We would like to thank Sheri Kelly, Derek Puchalski, Jeff
Van Doren, Patricia Boerckel, Joanne Erick, Dan Sylvester,
DeeMarie Skulsky, Christine Roberts, Amha Tadesse, Timothy Hamilton, Robert Wittrock, Michael Sharer, Elizabeth
Orlow Else, Liesje Germ, Mary Biersack, and Weli Li for their
expert technical assistance in performing the HPV serology
and PCR assays. We also thank Darcy Hille for assistance in
the preparation of this manuscript. Source of funding: Merck
Research Laboratories, a Division of Merck & Company
Inc., funded this study. Principal and main co-investigators:
Brazil—C Goes, G Andreoni, R Carneiro, E Fukazawa, J
Mesquita, F Coelho, M Perrotti; Finland—R Heikkila, R Zilliacus; Norway—JP Hoye, O-E Iversen, G Riis-Johannessen;
Sweden—A Andersson-Ellstrom, K Elfgren, G von Krogh;
USA—JT Comerci, RP Edwards, SA Gall, CM Peterson, YC
Wade.
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