Proof-of-Principle Evaluation of the Efficacy of Fewer Than Three Doses of a Bivalent HPV16/18 Vaccine
Three-dose regimens for human papillomavirus (HPV) vaccines are expensive and difficult to complete, especially in settings where the need for cervical cancer prevention is greatest. We evaluated the vaccine efficacy of fewer than three doses of the HPV16/18 vaccine Cervarix in our Costa Rica Vaccine Trial. Women were randomly assigned to receive three doses of the HPV16/18 vaccine or to a control vaccine and were followed for incident HPV16 or HPV18 infection that persisted in visits that were 10 or more months apart (median follow-up 4.2 years). After excluding women who had no follow-up or who were HPV16 and HPV18 DNA positive at enrollment, 5967 women received three vaccine doses (2957 HPV vaccine vs 3010 control vaccine), 802 received two doses (422 HPV vs. 380 control), and 384 received one dose (196 HPV vs. 188 control). Reasons for receiving fewer doses and other pre- and post-randomization characteristics were balanced within each dosage group between women receiving the HPV and control vaccines. Incident HPV16 or HPV18 infections that persisted for 1 year were unrelated to dosage of the control vaccine. Vaccine efficacy was 80.9% for three doses of the HPV vaccine (95% confidence interval [CI] = 71.1% to 87.7%; 25 and 133 events in the HPV and control arms, respectively), 84.1% for two doses (95% CI = 50.2% to 96.3%; 3 and 17 events), and 100% for one dose (95% CI = 66.5% to 100%; 0 and 10 events). Four years after vaccination of women who appeared to be uninfected, this nonrandomized analysis suggests that two doses of the HPV16/18 vaccine, and maybe even one dose, are as protective as three doses.
1444 Articles |JNCI Vol. 103, Issue 19 | October 5, 2011
DOI: 10.1093/jnci/djr319 © The Author(s) 2011. Published by Oxford University Press.
Advance Access publication on September 9, 2011. This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cervical cancer is the third most common cancer in women world-
wide, and it is the leading cause of cancer death among women in
some areas (1). Approximately 85% of cervical cancers occur in
developing countries without effective screening programs (1).
Administering prophylactic human papillomavirus (HPV) vaccines
to susceptible populations of young women could reduce a large
fraction of the disease burden.
The standard three-dose regimen of either Cervarix, the biva-
lent HPV16/18 vaccine with AS04 adjuvant (GlaxoSmithKline
Biologicals, Rixensart, Belgium), or Gardasil, the quadrivalent
HPV6/11/16/18 vaccine (Merck and Co, Whitehouse Station, NJ)
prevents HPV16 and HPV18 infections and related cervical
precancers among unexposed women (2,3). Currently, the cost of
these regimens and logistical difﬁculties associated with adminis-
tering three doses over 6 months make it impractical to vaccinate
preadolescent and young adult women in developing countries (4).
Even in developed countries, vaccine programs often do not suc-
cessfully administer all three doses; in the United States, a mi-
nority of vaccinees complete the full vaccine course (5); in
countries that have school-based health programs, such as Australia
(6) and the United Kingdom (7), vaccine uptake is higher. If vacci-
nation with fewer than three doses were to retain the high efﬁcacy
of the standard regimen, the ability to vaccinate more women for
the same cost could translate to a greater public health beneﬁt in
Women in our clinical trial in Costa Rica were randomly
assigned to receive three doses of either Cervarix or control
vaccine, yet approximately 20% received fewer than three doses
mostly because of pregnancy and referral to colposcopy. Here, we
compare the efﬁcacy of fewer than three doses of this HPV vaccine
vs the standard regimen to prevent newly detected persistent
HPV16 and HPV18 infections.
The 7153 women included in the present evaluation are among the
participants in an ongoing randomized clinical trial of 7466
women (8,9). The primary aim of the trial is to evaluate the
efficacy of a three-dose regimen of the Cervarix vaccine to prevent
persistent type-specific infection with HPV16 or HPV18 and the
Proof-of-Principle Evaluation of the Efficacy of Fewer Than
Three Doses of a Bivalent HPV16/18 Vaccine
Aimée R. Kreimer, Ana Cecilia Rodriguez, Allan Hildesheim, Rolando Herrero, Carolina Porras, Mark Schiffman, Paula González,
Diane Solomon, Silvia Jiménez, John T. Schiller, Douglas R. Lowy, Wim Quint, Mark E. Sherman, John Schussler,
Sholom Wacholder; for the CVT Vaccine Group
Manuscript received January 11, 2011; revised July 21, 2011; accepted July 22, 2011.
Correspondence to: Aimée R. Kreimer, PhD, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, 6120
Executive Blvd, EPS/7084, Rockville, MD 20852 (e-mail: email@example.com).
Background Three-dose regimens for human papillomavirus (HPV) vaccines are expensive and difficult to complete, espe-
cially in settings where the need for cervical cancer prevention is greatest.
Methods We evaluated the vaccine efficacy of fewer than three doses of the HPV16/18 vaccine Cervarix in our Costa Rica
Vaccine Trial. Women were randomly assigned to receive three doses of the HPV16/18 vaccine or to a control
vaccine and were followed for incident HPV16 or HPV18 infection that persisted in visits that were 10 or more
months apart (median follow-up 4.2 years). After excluding women who had no follow-up or who were HPV16
and HPV18 DNA positive at enrollment, 5967 women received three vaccine doses (2957 HPV vaccine vs 3010
control vaccine), 802 received two doses (422 HPV vs 380 control), and 384 received one dose (196 HPV vs 188
control). Reasons for receiving fewer doses and other pre- and post-randomization characteristics were
balanced within each dosage group between women receiving the HPV and control vaccines.
Results Incident HPV16 or HPV18 infections that persisted for 1 year were unrelated to dosage of the control vaccine.
Vaccine efficacy was 80.9% for three doses of the HPV vaccine (95% confidence interval [CI] = 71.1% to 87.7%;
25 and 133 events in the HPV and control arms, respectively), 84.1% for two doses (95% CI = 50.2% to 96.3%;
3 and 17 events), and 100% for one dose (95% CI = 66.5% to 100%; 0 and 10 events).
Conclusion Four years after vaccination of women who appeared to be uninfected, this nonrandomized analysis suggests
that two doses of the HPV16/18 vaccine, and maybe even one dose, are as protective as three doses.
J Natl Cancer Inst 2011;103:1444–1451
jnci.oxfordjournals.org JNCI |Articles 1445
subsequent development of HPV-associated precancerous lesions
In June 2004 and December 2005, the study enrolled young
women who resided in the regions of Guanacaste and Puntarenas,
Costa Rica, and were identiﬁed via a census. To be eligible, women
were required to be 18–25 years of age, in good general health,
and neither pregnant nor breastfeeding. Women were excluded
if they had a preexisting medical condition that would preclude
vaccination, a history of hepatitis A infection or previous vaccina-
tion against hepatitis A, or if they were unwilling to use contra-
ception during the vaccination period. The trial was approved by
human subjects review committees of the US National Cancer
Institute and Instituto Costarricense de Investigación y Enseñanza
en Nutrición y Salud (INCIENSA) in Costa Rica. In the United
States, it was registered as Clinical Trial number NCT00128661.
At the enrollment visit and following informed consent, a risk
factor interview was administered, a pelvic examination was
performed on sexually experienced women, exfoliated cells were
collected in PreservCyt liquid medium (Cytyc Corp, now Hologic,
Marlborough, MA) for Thinprep (Cytyc Corp) cytological evalua-
tion and HPV DNA testing, and blood was collected (for 90% of
women, 30 mL; for the 10% of women in our immunogenicity
subcohort, 70 mL). Then, women were randomly assigned
in a double-blinded fashion to receive either Cervarix or a
“control” hepatitis A vaccine (a modiﬁed preparation of Havrix;
GlaxoSmithKline Biologicals) (0.5 mL per dose). Both vaccines
had identical packaging and were intended to be administered at 0, 1,
and 6 months. At the 6-month vaccination visit, sexually experi-
enced women self-collected a cervicovaginal exfoliated cell spec-
imen for HPV DNA testing. Women who were not vaccinated in
the allowable time frames (ie, 21–120 days and 121–300 days after
enrollment for doses two and three, respectively) did not receive
the dose. Women who became pregnant during the vaccination
phase or who were referred to colposcopy were deferred, so that
they missed the dose if the vaccination window was missed. A Data
Safety Monitoring Board reviewed safety data annually during the
vaccination phase and as needed during the follow-up period (ﬁnal
review: November 10, 2010).
The protocol required all women to be seen each year during
the 4 years of follow-up. At each annual study visit, clinicians col-
lected exfoliated cells from sexually active women for cytological
evaluation and HPV DNA testing. Women found to have low-
grade squamous intraepithelial neoplasia (LSIL) or HPV-positive
atypical squamous cells of undetermined signiﬁcance (ASCUS)
underwent the same procedures at 6-month intervals for safety
until three consecutive normal cytological results, when they
returned to yearly follow-up. Women with evidence of high-
grade disease or persistent low-grade abnormalities were
referred to colposcopy for evaluation and treatment if needed.
After the 4-year visit, a modiﬁed algorithm for colposcopic
referral, biopsy, and treatment was applied to assure safety
of participants at the completion of the initial 4-year study
Cervical samples were shipped from the clinic to the laboratory
in Costa Rica in controlled temperature coolers. Duplicate 0.5 mL
aliquots were made for HPV DNA testing by polymerase chain
reaction (PCR), and they were frozen in liquid nitrogen. These
samples were stored frozen in the repository in Costa Rica and
then shipped in frozen batches to the Netherlands for HPV DNA
PCR-based HPV DNA testing was performed using the SPF10
PCR primer system and a DNA enzyme immunoassay detection of
amplimers (DEIA; DDL Diagnostic Laboratory, Voorburg, the
Netherlands) (11). Brieﬂy, 10 µL proteinase K–treated DNA was
added to 40 µL of PCR mix. The SPF10 PCR primer set ampliﬁes
a small fragment of 65 bp from the L1 region of mucosal HPV
genotypes. Ampliﬁcation products were detected using the HPV
SPF10 PCR DEIA system. DEIA-positive SPF10 amplimers were
used to identify the HPV genotype by reverse hybridization with
the HPV line probe assay (LiPA25), containing probes for 25
different HPV genotypes (HPV genotypes 6, 11, 16, 18, 31, 33,
34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73,
70, and 74; SPF10 HPV LiPA25 version 1 [Labo Biomedical
Products, Rijswijk, the Netherlands, based on licensed Innogenetics
technology]) (12). To ensure that HPV16 and HPV18 infections
were not missed, all specimens that were positive for the presence
of HPV DNA using the SPF10 DEIA assay but negative for pres-
ence of HPV16 or HPV18 by the LiPA25 assay were also tested
with type-speciﬁc PCR primer sets used to selectively amplify a
92 bp HPV16 E7 fragment (TS16) and a 126 bp HPV18 L1
fragment (TS18). Amplimers from the type-speciﬁc PCRs were
detected by DEIA, similar to the method for SPF10 amplimer
CONTEXT AND CAVEATS
The HPV16/18 vaccine Cervarix is normally given in three doses.
Previously, there were no efficacy data to establish whether fewer
doses might protect women against cervical cancer.
Data were taken from the Costa Rica vaccine trial, in which many
of the 7153 women missed one or more of three prescribed doses
of a randomly assigned HPV16/18 vaccine or control (hepatitis A)
vaccine mostly because of pregnancy and referral to colposcopy.
Vaccine efficacy was evaluated in each dosage group by determi-
nation, via HPV DNA testing, of the number of newly detected
HPV16 or HPV18 infections that persisted at least 1 year.
The estimated vaccine efficacy against infection with HPV16 and
HPV18 was similar whether the woman received one, two, or all
It appears that two doses, or even one dose, of the HPV16/18 vac-
cine, is highly efficacious in protecting against persistent HPV16/18
It is still not known whether the three-dose regimen might provide
longer duration of protection or more cross-protection against
heterologous HPV types and whether the findings in this trial are
applicable to populations in other geographical settings or to other
From the Editors
1446 Articles |JNCI Vol. 103, Issue 19 | October 5, 2011
Results follow a statistical analysis plan that was prepared before the
investigation of this question was initiated. Fieldwork is ongoing,
and individual information remains blinded. Analyses were therefore
conducted by an external group, Information Management Systems
(Rockville, MD), under the direction of the investigators who
remained masked to individual random assignments.
All women were included in the analysis except those who were
both HPV16 and HPV18 DNA positive at enrollment or had no
follow-up visits post-enrollment. Women in the HPV and control
arms were grouped according to the number of doses they received.
Reasons for not receiving the full-dosing regimen were compared
by use of the x2 test for categorical variables. Median follow-up
time from enrollment was calculated in months by arm and dose
and compared by use of the nonparametric Kruskal–Wallis test.
The primary endpoint for this analysis was a newly detected
HPV16 or HPV18 infection that persisted for at least 10 months,
which is an intermediate cancer endpoint that is associated with
development of cervical intraepithelial neoplasia 3 (14). The def-
inition of that endpoint (persistent infection) required two de-
tections of infection by the same HPV type that occurred at least
10 months apart with no intervening negative tests. We required
the ﬁrst detection of the infection to be at the 6-month vaccine
visit or later to avoid misclassiﬁcation of infections prevalent at
enrollment. In this woman-level analysis, each woman could
only contribute once to the numerator (number of women who
had an incident persistent HPV16 and/or HPV18 infection) and
denominator (all women with at least one study visit post-enroll-
ment and HPV 16 and/or HPV18 DNA negative at enrollment),
even if multiple HPV types were detected. Furthermore, HPV in-
fection status was assessed at both regular and colposcopy study
We evaluated 6-month persistence as a secondary endpoint
because it is more distal to precancer; 6-month persistence was
deﬁned as two or more positive tests for a given HPV type that
occurred at least 4 months apart with no intervening negative tests.
We also assessed VE for incident 12-month persistent HPV31,
HPV33, and HPV45 infections combined because these are the
HPV types for which there is prior evidence of vaccine cross-
protection (2,10); in this analysis, we excluded women with prevalent
HPV31, HPV33, and HPV45 infections detected at enrollment.
The endpoint for this analysis was deﬁned as a new type-speciﬁc
HPV31, HPV33, or HPV45 infection that was ﬁrst detected at the
6-month vaccine visit or later, and again at a visit at least 10 months
later, with no intervening negative tests for the HPV type in
For each dose group and arm, we deﬁned the attack rate as the
proportion of the number of events among the number of women
over the 4 years of the study. Any difference in the attack rates
among women who received one, two, or three doses of the
control vaccine could reﬂect random variation or underlying
differences in the risk of incident infection by the number of doses;
the absence of a difference in the attack rates in the control vaccine
arm (in which women are not protected from HPV infections
unlike the HPV vaccine arm) would indicate that determinants of
risk, like sexual behavior, are not biasing the comparison between
number of doses received.
Within each dosage group, the complement of the ratios of the
attack rates for the HPV and the control arms are the VE estimates.
We used data from the control arm, instead of directly comparing
by the number of doses within the HPV arm alone, because we were
not certain whether the risk of a new persistent HPV16 or HPV18
infection might vary by number of doses received, even if no HPV
vaccine had been administered. We calculated exact conﬁdence
intervals (15) for VE based on the binomial distribution of the
number of events in the HPV arm among the total number of events
in the HPV and control arms. The exact conﬁdence limits for VE
use numerators of the attack rates based on the product of the total
number of events and the exact binomial limit (16).
The VE estimate for three doses is presented in this article
to compare that for two doses and one dose. The three-dose VE
estimate in the current analysis, however, differs from our previ-
ously presented estimate (90.9% for according to protocol efﬁcacy)
(10) because of slightly different analytical cohorts: the present
analysis did not exclude based on disease, treatment, or vaccination
windows, and it counted outcomes starting at the 6-month visit
instead of the 12-month visit.
If we found statistically signiﬁcant evidence (ie, a 95% CI ex-
cluding zero) that two or one dose(s) of vaccine conferred VE, we
calculated the ratios of VEs for two vs three doses and for one vs three
doses and their corresponding 95% conﬁdence intervals using an
unconditional bootstrap percentile method (17) with 1000 bootstrap
datasets, where resampling was done with replacement. Because the
cost of administering a series of vaccinations is approximately propor-
tional to the number of doses given, we prespeciﬁed that a ratio of 2:3
for the VEs of HPV in women who received two and three doses
would reﬂect equal efﬁcacy per dose and per cost. Thus, we would
claim that vaccination with fewer than three doses was successful if we
rejected a one-sided test of the null hypothesis that the ratio of
the two-dose and three-dose VEs equals two-thirds, equivalent to the
lower bound of the 95% conﬁdence interval for the estimate of the
ratio being above two-thirds. For one dose, the corresponding crite-
rion for claiming a positive result is that the lower bound of the 95%
conﬁdence interval for the estimate of the ratio is above one-third.
The 12 months of persistence required for the endpoint began
with an incident infection at the 6-month visit, when the ﬁnal
vaccine dose was scheduled to be administered, or later. A sensi-
tivity analysis that included only infections incident at the
12-month visit or later as endpoints addressed the possibility of
bias from differential assessment of outcomes in women who
missed vs received the 6-month vaccination.
The present analysis contains updated data until June 21, 2010,
when the analytical database was frozen; using the available data at
the initial January 1, 2010 data lock point, statistically signiﬁcant
vaccine efﬁcacy (VE) was observed by dose (VE for three doses =
78%, 95% conﬁdence interval [CI] = 67% to 86%; VE for two
doses = 82%, 95% CI = 43% to 96%; VE for one dose = 100%,
95% CI = 57% to 100%).
Of the 24 467 women who we screened, 7466 women were enrolled
and were randomly assigned to the HPV16/18 vaccine Cervarix or
to a control vaccine for hepatitis A (Figure 1). We excluded women
jnci.oxfordjournals.org JNCI |Articles 1447
who had no post-enrollment visits or who were HPV16 and
HPV18 DNA positive at enrollment (155 in the HPV vaccine arm;
158 in the control vaccine arm); the analytic cohort comprised
5967 women who received three doses (of whom 2957 received the
HPV vaccine and 3010 received the control vaccine), 802 women
who received two doses (of whom 422 received the HPV and 380
received the control vaccine), and 384 women who received one
dose (of whom, 196 received the HPV and 188 received the con-
trol vaccine) (P for differences in the number of women receiving
the HPV vaccine vs the control vaccine by dose = 0.23). Median
time of follow-up from date of first vaccine administration was
50 months (4.2 years) and was similar between arms within dose.
Participants’ reasons for not receiving all doses were similar in
both arms conditional on the number of doses actually received
(P > .05 for all comparisons; Table 1). The most common reasons
for not receiving all three doses were involuntary, including preg-
nancy and colposcopy referral; it was less common for participants
to refuse the vaccine.
For all dosage groups, women who received the HPV vaccine
vs the control vaccine were comparable with respect to age at entry
and total number of visits (Table 2). Among those who received
two doses or one dose of the vaccine, HPV16 and HPV18 DNA
and serologic status at enrollment were comparable by arm within
each dose group. Among women who received three doses, we
observed no differences in serologic status at enrollment; however,
women in the control group were marginally more likely to have
been HPV16 and/or HPV18 DNA positive at enrollment than
women who received the HPV vaccine (8.9% vs 7.5%, P = .05), as
noted previously (8,9). In the control arm, the attack rates of inci-
dent HPV16 or HPV18 infections that persisted for 1 year were
Table 1. Reasons for missing doses among women who received two doses or one dose of vaccine*
Reason for missed dose
Missing second or third dose,
among women who received
Missing second dose,
among women who received
Missing third dose, among
women who received
HPV, No. (%) Control, No. (%) HPV, No. (%) Control, No. (%) HPV, No. (%) Control, No. (%)
Pregnancy 155 (31.8) 145 (32.9) 35 (12.6) 35 (12.8) 50 (18.1) 57 (20.8)
Colposcopy referral 13 (2.7) 12 (2.7) 57 (20.6) 45 (16.4) 57 (20.6) 42 (15.3)
Medical condition 79 (16.2) 66 (15.0) 49 (17.7) 60 (21.9) 43 (15.5) 57 (20.8)
Vaccine refusal by participant 63 (12.9) 58 (13.2) 42 (15.2) 38 (13.9) 87 (31.4) 84 (30.7)
Missed study visit 98 (20.1) 96 (21.8) 58 (20.9) 55 (20.1) 20 (7.2) 23 (8.4)
Other‡ 80 (16.4) 64 (14.5) 36 (13.0) 41 (15.0) 20 (7.2) 11 (4.0)
* This table includes all vaccinated women to prevent unblinding that could happen with cells that had small sample size (ie, fewer than five women). HPV =
† For women who received only one dose, it was possible to have different reasons for missing each of the two subsequent doses.
‡ The three most common “other” reasons included: the woman could not get time off from work to come into the clinic for a vaccination, personal reasons, or
the woman was not using an acceptable form of birth control.
24,467 Women screened during
7466 Women randomized*
# of incident 1 year–persistent
Women who received 3 doses
Women who received 2 doses
Women who received 1 dose
# of incident 1 year–
# of incident 1 year–
3561 ineligible (out of area)
2186 ineligible (other)
1527 not located
4569 deferred status at end of
Figure 1. CONSORT diagram of women in
the Costa Rica Vaccine Trial. The primary
aim of the trial was to evaluate the efﬁcacy
of a three-dose regimen of the Cervarix
vaccine to prevent persistent type-speciﬁc
infection with HPV16 or HPV18 and the
subsequent development of HPV-associated
precancerous lesions. Although 7466
women were randomized to receive three
doses of either Cervarix or control vaccine,
approximately 20% received fewer than
three doses of Cervarix or control vaccine
mostly because of pregnancy and referral to
colposcopy. Thus, we were able to investi-
gate the protection afforded by two and one
dose(s) of the HPV vaccine because the cost
and logistical difﬁculties of the standard
three-dose vaccine regimen compromises
implementation. Asterisk indicates that
four women received discordant vaccines
(one woman was enrolled twice and
received three doses of each vaccine and
three women received two doses of one
vaccine and one dose of the other vaccine).
For the purpose of this analysis, the control
dosing was ignored and they were catego-
rized based on the number of HPV vaccines
they received. Dagger indicates that women
who were both HPV16 and HPV18 DNA
positive at enrollment were excluded,
as were women with no follow-up visits
1448 Articles |JNCI Vol. 103, Issue 19 | October 5, 2011
similar among women who received three doses (4.4%), two doses
(4.5%), or one dose (5.3%) indicating that they were at similar risk
for acquiring HPV infections regardless of the number of doses
received (Table 3).
VE for three doses against newly detected HPV16 or HPV18
that persisted at least 1 year was 80.9% (95% CI = 71.1% to
87.7%; 25 and 133 events in the HPV and control arms, respec-
tively), for two doses was 84.1% (95% CI = 50.2% to 96.3%; three
and 17), and for one dose was 100% (95% CI = 66.5% to 100%;
zero and 10); no statistically signiﬁcant trend for increasing VE
with fewer doses was observed (Ptrend = .21) (Table 3). VE results
were similar for the 6-month persistent HPV16 and/or HPV18
Table 2. Participant characteristics by number of vaccine doses received and vaccine arm*
One dose† Two doses† Three doses†
HPV, No. (%) Control, No. (%) HPV, No. (%) Control, No. (%) HPV, No. (%) Control, No. (%)
Age at entry, y‡
18–19 53 (27.0) 52 (27.7) 140 (33.2) 143 (37.6) 933 (31.6) 982 (32.6)
20–21 56 (28.6) 49 (26.1) 117 (27.7) 85 (22.4) 739 (25.0) 725 (24.1)
22–23 42 (21.4) 40 (21.3) 90 (21.3) 80 (21.1) 661 (22.4) 704 (23.4)
24–25 45 (23.0) 47 (25.0) 75 (17.8) 72 (18.9) 624 (21.1) 599 (19.9)
No. of clinic visits attended§
1–2 13 (6.6) 17 (9.0) 36 (8.5) 37 (9.7) 74 (2.5) 68 (2.3)
3–5 96 (49.0) 99 (52.7) 275 (65.2) 224 (58.9) 1912 (64.7) 1867 (62.0)
6–8 67 (34.2) 62 (33.0) 97 (23.0) 106 (27.9) 813 (27.5) 896 (29.8)
≥9 20 (10.2) 10 (5.3) 14 (3.3) 13 (3.4) 158 (5.3) 179 (5.9)
Median (IQR) 5 (4 to 7) 5 (4 to 7) 5 (4 to 6) 5 (4 to 6) 5 (5 to 6) 5 (5 to 6)
HPV16/18 DNA status at enrollment||
Negative¶ 245 (88.4) 241 (88.0) 438 (89.8) 403 (91.4) 2742 (92.5) 2751 (91.1)
Positive# 32 (11.6) 33 (12.0) 50 (10.2) 38 (8.6) 223 (7.5)** 270 (8.9)
HPV16/18 serostatus at enrollment||
Negative 173 (62.5) 165 (60.2) 294 (60.2) 269 (61.0) 1899 (64.0) 1899 (62.9)
Positive# 104 (37.5) 109 (39.8) 194 (39.8) 172 (39.0) 1066 (36.0) 1122 (37.1)
* IQR = interquartile range; HPV = human papillomavirus.
† The women who received discordant vaccines were categorized according to the number of HPV vaccine doses they received.
‡ Two women enrolled at age 17 years were included in the 18–19 year age group; one woman enrolled at age 26 and one woman enrolled at age 27 were in-
cluded in the 24–25 year age group.
§ Includes clinic visits for vaccination, annual screening, colposcopy, and treatment (when needed).
|| Data on HPV16 and HPV18 status at enrollment include all vaccinated women to prevent unblinding that could happen with cells that had small sample
size (ie, fewer than five women).
¶ Included in the negative category are virgins (who did not provide a cervical specimen for HPV testing) and three women who were missing enrollment HPV
polymerase chain reaction results
# Indicates positive for either or both HPV16 and HPV18 at enrollment.
** Women in the control group who received three doses were marginally more likely to be HPV16 and/or HPV18 DNA positive at enrollment compared with
women who received three doses of the HPV vaccine (8.9% vs 7.5%, P = .05 using two-sided test of equality of proportions), as noted previously (8,9).
Table 3. Estimated vaccine efficacy against 12-month incident persistent infection for women who received one, two, and three doses
of a HPV vaccine compared with a control vaccine
Doses, No. Arm
Proportion of women with incident,
12-month persistent HPV16 or
HPV18 infections, % (95% CI)*
Efficacy relative to
% (95% CI)*
Control 3010 133 4.4% (3.7% to 5.2%) 80.9% (71.1% to 87.7%) Referent
HPV 2957 25 0.85% (0.56% to 1.2%)
2‡ Control 380 17 4.5% (2.7% to 6.9%) 84.1% (50.2% to 96.3%) 104% (69.3% to 129%)
HPV 422 3 0.71% (0.18% to 1.9%)
1 Control 188 10 5.3% (2.7% to 9.3%) 100% (66.5% to 100%) 124%§
HPV 196 0 0.0% (0.0% to 1.5%)
* Human papillomavirus = HPV; 95% CI = 95% confidence interval.
† The distribution of the time at diagnosis of the case patients in the HPV and control arms was qualitatively assessed to determine whether the protection
afforded by two doses may be short lived compared with that of three doses. Twenty (80.0%) of 25 breakthrough 1-year persistent HPV infections in the vaccine
arm were first detected in the first year of follow-up (suggesting missed prevalent infections at enrollment) compared with 40 (30.1%) of 133 infections detected
in the control arm. Sixteen (64.0%) of 25 breakthrough infections occurred among women who were HPV16 seropositive at enrollment.
‡ One of the three breakthrough infections was detected in each of the first 3 years of the study compared with 0%, 64.7%, 23.5%, and 11.8% of the 17 infec-
tions in years 1, 2, 3, and 4 of the study, respectively. One (33.3%) of the three breakthrough infections occurred in a woman who was HPV16 seropositive at
§ No bootstrap confidence interval could be estimated due to the presence of zero events in the HPV arm after one dose of vaccine.
jnci.oxfordjournals.org JNCI |Articles 1449
infection endpoint (Supplementary Table 1, available online).
Although we did see cross-protection against incident 1-year per-
sistent infection with HPV31, HPV33, and HPV45 combined in
women who received the standard three-dose regimen (41.3%
[95% CI = 18.9% to 57.9%]; 57 and 99 events in the HPV and
control arms, respectively) (8), there was no evidence of such cross-
protection in women who received two doses: 225.9% (95%
CI = 2334% to 61.1%; seven and ﬁve). The small number of total
events (n = 7) limited our ability to evaluate cross-protection
among women who received only one dose.
Because there was statistically signiﬁcant evidence that two doses
or even one dose of the vaccine had VE against HPV16 and HPV18
(ie, for all VEs, the lower bound of the 95% CI was substantially
greater than 0%), we computed the ratios of the VEs for fewer
than three doses vs full regimen. The ratio of 2:3 dose VEs was
104% (95% CI = 69.3% to 129%), consistent with two doses
being more than two-thirds as effective as three doses (Table 3).
The 1:3 ratio of VEs was 124%, but no bootstrap conﬁdence
interval could be estimated because of the absence of events in
the HPV arm.
In the sensitivity analysis that investigated the potential bias
from differential assessment of outcomes by dose, VEs excluding
the 6-month study visit were comparable: 82.4% (95% CI = 72.7%
to 89.0%) for three doses, 84.1% (95% CI = 50.2% to 96.3%) for
two doses, and 100% (95% CI = 66.5% to 100%) for one dose.
We investigated the protection afforded by two and one dose(s) of
the HPV vaccine because the cost and logistical difficulties of the
standard three-dose vaccine regimen compromises implementa-
tion of this life-saving measure in resource-poor settings. Nested
within our phase III randomized clinical trial in Costa Rica, we
provide the first clinical evidence that two doses of the bivalent
HPV vaccine are highly efficacious in the prevention of incident
HPV16 and HPV18 infections that persist for at least 1 year
(VE = 84.1% [95% CI = 50.2% to 96.3%]), and that even a single
dose may be highly efficacious (VE = 100% [95% CI = 66.5%
Although this analysis was not randomized, the attack rates of
new infections were essentially equal among women who received
one, two, and three doses of the control vaccine. Equal attack rates
in the control arm suggested that risks of infection were the same
regardless of number of doses received. Furthermore, pregnancy,
the most common reason that women received a reduced number
of doses, was unrelated to vaccine assignment (18) and therefore
unlikely to bias the VE estimates. These observations provide sup-
port for the robustness of our analysis.
Further evaluation of the efﬁcacy of one dose is particularly
important for several reasons. The protection afforded by a single
dose of vaccine was unexpected because other subunit vaccines
typically require boosting following the prime dose to confer long-
term protection, although perhaps many of our sexually active
participants had been previously exposed to HPV16 or HPV18.
Also, the number of events (N = 10 for ≥12-month persistent
infection; N = 15 for ≥6-month persistent infection) was small, and
follow-up time was limited.
Available evidence from immunologic studies supports our efﬁ-
cacy ﬁndings. Girls who received two and three doses of Gardasil
had similar antibody titers several years post-vaccination (19).
Women in the control arm of our trial with relatively high anti-
body titers after natural infection were partially protected against
subsequent incident HPV16 and HPV18 infection (20). However,
the exact relationship between these antibody markers and future
risk of disease is not clear.
Important questions remain unanswered. The full three-dose reg-
imen may confer greater cross-protection (2,10) against heterologous
HPV types, as suggested by the limited evidence here. Additionally,
data from a trial of the bivalent HPV16/18 vaccine in Costa Rica may
not apply to other vaccine formulations (such as the quadrivalent
HPV vaccine with alum adjuvant) or to other populations who may
have more comorbidities than our study population (eg, endemic
parasitic infections that could, in turn, produce malnutrition and
possibly a less robust immune response). Finally, the duration of
protection for fewer than three doses must be quantiﬁed. Future
studies, such as the cluster-randomized trial that is being con-
ducted in India by the World Health Organization, are the next
critical step in addressing these important research questions.
Our clinical efﬁcacy data provide suggestive evidence that an
HPV vaccine program that could vaccinate 50% more women with
a two-dose regimen could potentially reduce cervical cancer inci-
dence more than a standard three-dose program that uses the same
number of total doses but in fewer women. Surveillance of the
ongoing programs with extended intervals (vaccination at 0, 6, and
60 months), such as those currently implemented in Quebec and
Mexico, can monitor the short-term effectiveness of administering
fewer than three doses. If randomized studies and cost-effectiveness
analyses conﬁrm the net beneﬁts of administering fewer doses, and
the duration of protection is sufﬁcient, then the need for fewer
doses may make primary prevention of cervical cancer a reality,
especially for women in areas where most cervical cancers occur.
1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates
of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer.
2. Paavonen J, Naud P, Salmeron J, et al. Efﬁcacy of human papillomavirus
(HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and pre-
cancer caused by oncogenic HPV types (PATRICIA): ﬁnal analysis of a
double-blind, randomised study in young women. Lancet. 2009;374(9686):
3. FUTURE II Study Group. Quadrivalent vaccine against human papillo-
mavirus to prevent high-grade cervical lesions. N Engl J Med. 2007;356(19):
4. Goldie SJ, O’Shea M, Diaz M, Kim SY. Beneﬁts, cost requirements and
cost-effectiveness of the HPV16,18 vaccine for cervical cancer prevention
in developing countries: policy implications. Reprod Health Matters. 2008;
5. Centers for Disease Control and Prevention. National, state, and local
area vaccination coverage among adolescents aged 13–17 years—United
States, 2008. MMWR Morb Mortal Wkly Rep. 2009;58(36):997–1001.
6. Immunise Australia Program. Human Papillomavirus (HPV). http://www.
immunise-hpv. Accessed July 12, 2011.
documents/digitalasset/dh_123826.pdf. Accessed July 12, 2011.
8. Herrero R, Hildesheim A, Rodriguez AC, et al. Rationale and design of a
community-based double-blind randomized clinical trial of an HPV 16
1450 Articles |JNCI Vol. 103, Issue 19 | October 5, 2011
and 18 vaccine in Guanacaste, Costa Rica. Vaccine. 2008;26(37):
9. Hildesheim A, Herrero R, Wacholder S, et al. Effect of human papilloma-
virus 16/18 L1 viruslike particle vaccine among young women with preex-
isting infection: a randomized trial. JAMA. 2007;298(7):743–753.
10. Herrero R, Wacholder S, Rodríguez AC, et al. Prevention of persistent
HPV infection by a HPV 16–18 vaccine: a community based randomized
clinical trial in Guanacaste, Costa Rica. Oral presentation #459, presented
at the 25th International Papillomavirus Conference; July 3-8, 2010.
11. Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-fragment PCR
assay for highly sensitive broad-spectrum detection of anogenital human
papillomaviruses. Am.J Pathol. 1998;153(6):1731–1739.
12. Kleter B, van Doorn LJ, Schrauwen L, et al. Development and clinical
evaluation of a highly sensitive PCR-reverse hybridization line probe assay
for detection and identiﬁcation of anogenital human papillomavirus. J Clin
13. van Doorn LJ, Molijn A, Kleter B, Quint W, Colau B. Highly effective
detection of human papillomavirus 16 and 18 DNA by a testing algorithm
combining broad-spectrum and type-speciﬁc PCR. J Clin Microbiol.
14. Koshiol J, Lindsay L, Pimenta JM, et al. Persistent human papillomavirus
infection and cervical neoplasia: a systematic review and meta-analysis. Am
J Epidemiol. 2008;168(2):123–137.
15. Agresti A. Categorical Data Analysis. 2nd ed.. Hoboken, NJ: John Wiley
and Sons, Inc; 2002.
16. Rothman KJ, Boice JD. Epidemiologic Analysis with a Programmable
Calculator. New Edition. Boston, MA: Epidemiology Resources, Inc; 1982.
17. Efron B. The Jackknife, the Bootstrap and Other Resampling Plans.
Philadelphia, PA: Society for Industrial and Applied Mathematics; 1982.
18. Wacholder S, Chen BE, Wilcox A, et al. Risk of misscarriage with bivalent
vaccine against human papillomavirus (HPV) types 16 and 18: pooled
analysis of two randomised controlled trials. BMJ. 2010;340:c712.
19. Dobson S, Dawar M, Kollman T, et al. A two dose HPV vaccine schedule
in girls: immunogenecity at 24 months. Poster #691, presented at the 25th
International Papillomavirus Conference. July 3-8, 2010.
20. Safaeian M, Porras C, Schiffman M, et al; Costa Rican Vaccine Trial
Group. Epidemiological study of anti-HPV 16/18 seropositivity and
subsequent risk of HPV 16 and -18 infections. J Natl Cancer Inst.
The Costa Rica HPV Vaccine Trial is a long-standing collaboration between
investigators in Costa Rica and the US National Cancer Institute (NCI). The
trial is sponsored and funded by the NCI (contract N01-CP-11005), with funding
support from the National Institutes of Health Ofﬁce of Research on Women’s
This study was conducted with the support from the Ministry of Health of
Costa Rica. Vaccine was provided for our trial by GlaxoSmithKline Biologicals
(GSK), under a Clinical Trials Agreement with the NCI. GSK also provided
support for aspects of the trial associated with regulatory submission needs of
the company under FDA BB-IND 7920. SchillerJ. T. Schiller and D. R. Lowy
report that they are named inventors on US government–owned HPV vaccine
patents that are licensed to GSK and Merck and for which the NCI receives
licensing fees. J. T. Schiller and D. R. Lowy are entitled to limited royalties as
speciﬁed by federal law. No other ﬁnancial disclosures were reported.
The NCI and Costa Rica investigators are responsible for the design and
conduct of the study; collection, management, analysis, and interpretation of
the data; and preparation of the article. The NCI and Costa Rica investigators
make ﬁnal editorial decisions on this and subsequent publications; GSK has the
right to review and comment.
Cervarix is a registered trademark of the GlaxoSmithKline group of
ARK, SW, ACR, AH, RH, CP, DS, and MS designed the analysis. JS con-
ducted all statistical programming under direction of ARK and SW. PW, SW,
ACR, AH, RH, CP, DS, MS, MES, and SJ were responsible for data collection.
WQ was responsible for all HPV-related test results. ARK, SW, ACR, AH,
RH, CP, DS, and MS analyzed the data. AKR, SW, ACR, AH, RH, CP, DS,
MS, DRL, and JTS interpreted the data. ARK and SW wrote the article. ARK,
SW, ACR, AH, RH, DS, MS, DRL, JTS, MES, and JS critically reviewed all
material for important intellectual content. ARK and SW are the guarantors of
all material contained herein.
The names and afﬁliations of investigators in the Costa Rica Vaccine Trial
(CVT) group are given below.
Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José,
Costa Rica: Mario Alfaro (Cytopathologist); Manuel Barrantes (Field Supervisor);
M. Concepción Bratti (co-Investigator); Fernando Cárdenas (General Field
Supervisor); Bernal Cortés (Specimen and Repository Manager); Albert
Espinoza (Head, Coding and Data Entry); Yenory Estrada (Pharmacist); Paula
González (co-Investigator); Diego Guillén (Pathologist); Rolando Herrero
(co-Principal Investigator); Silvia E. Jiménez (Trial Coordinator); Jorge
Morales (Colposcopist); Luis Villegas (Colposcopist); Lidia Ana Morera (Head
Study Nurse); Elmer Pérez (Field Supervisor); Carolina Porras (co-Investiga-
tor); Ana Cecilia Rodríguez (co-Investigator); Libia Rivas (Clinical coordinator).
University of Costa Rica, San José, Costa Rica: Enrique Freer (Director, HPV
Diagnostics Laboratory); José Bonilla (Head, HPV Immunology Laboratory);
Alfonso García-Piñeres (Immunologist); Sandra Silva (Head Microbiologist,
HPV Diagnostics Laboratory); Ivannia Atmella (Microbiologist, Immunology
Laboratory); Margarita Ramírez (Microbiologist, Immunology Laboratory).
US National Cancer Institute, Bethesda, MD: Allan Hildesheim (co-Prin-
cipal Investigator & NCI co-Project Ofﬁcer); Aimée R. Kreimer (Investigator);
Douglas R. Lowy (HPV Virologist); Nora Macklin (Trial Coordinator); Mark
Schiffman (Medical Monitor & NCI co-Project Ofﬁcer); John T. Schiller
(HPV Virologist); Mark Sherman (QC Pathologist); Diane Solomon (Medical
Monitor & QC Pathologist); Sholom Wacholder (Statistician).
SAIC, NCI-Frederick, Frederick, MD: Ligia Pinto (Head, HPV Immunology
Laboratory); Troy Kemp (Scientist, HPV Immunology Laboratory).
Women’s and Infants’ Hospital, Providence, RI: Claire Eklund (QC Cytology);
Martha Hutchinson (QC Cytology).
Georgetown University, Washington, DC: Mary Sidawy (Histopathologist).
DDL Diagnostic Laboratory, the Netherlands: Wim Quint (Virologist,
HPV DNA Testing); Leen-Jan van Doorn (HPV DNA Testing).
We would like to extend a special thanks to the women of Guanacaste and
Puntarenas, Costa Rica, who gave of themselves in participating in this effort.
We also acknowledge the tremendous effort and dedication of the staff in Costa
Rica involved in this project, including Bernardo Blanco and his team (census);
Ricardo Cerdas and Ana Hernández (blood processing); José Miguel González,
Osman López, Johnny Matamoros, Manuel Sánchez, Rafael Thompson, and
Jorge Umaña (ﬁeld activity coordinators); Su Yen Araya, Hazel Barquero,
Hayleen Campos, Muriel Grijalba, Ana Cristina Monge, Ana Peraza, Diana
Robles, María Fernanda Sáenz, Dorita Vargas, and Jessica Vindas (clinic
coordinators); Paola Alvarez, Dinia Angulo, Ana Live Arias, Betzaida Barrantes,
Marianela Bonilla, Mary José Calvo, Loretto Carvajal, Jessenia Chinchilla,
Blanca Cruz, Marianela Herrera, Andrea Interiano, Fabiola Jiménez, Erick
Lagos, Viviana Loría, Andrea Messeguer, Rebeca Ocampo, Silvia Padilla,
Angie Ramírez, Libia Rivas, Daniela Romero, Byron Romero, Jessenia Ruiz,
Daniela Ruiz, Genie Saborío, Sofía Ssoto, Malena Salas, Adriana Torrez,
Natalia Ugalde, Ana Cristina Ugalde, Adriana Vallejos, Yesenia Vázquez,
Maricela Villegas (clinicians); Marta Alvarado, Ana Cristina Arroyo, Gloriana
Barrientos, Diana Díaz, Marlen Jara, Maureen Matarrita, María Ester Molina,
Elida Ordóñez, Gina Sánchez, and Zihara Villegas (nurses); Arianne Castrillo
and Vivian López (education and outreach effort coordinators); Karla Coronado
(appointment coordinator); Ricardo Alfaro (quality control coordinator);
Charles Sánchez and Livia Romero (document center coordinators); Cristian
Montero (quality assurance, regulatory); and Carlos Avila and Eric Alpízar (IT
coordinators). Special recognition is also extended to Sofía Elizondo, Executive
Director of Fundación INCIENSA and her staff for their administrative sup-
port. In the United States, we would like to extend our appreciation to the team
from Information Management Services (IMS) responsible for the develop-
ment and maintenance of the data system used in the trial and who serve as the
data management center for this effort. We would like to speciﬁcally acknowl-
edge the invaluable contributions made by Jean Cyr, Julie Buckland, Laurie
Rich, Brian Befano, and Dennis Buckman. We acknowledge the contributions
made by individuals at Westat, Inc, who provided project development and/
or monitoring support, including Kerry Grace Morrisey, Kirk Midkiff, Susan
jnci.oxfordjournals.org JNCI |Articles 1451
Truitt, Sonia Stoszek, Maribel Gomez, and Isabel Trejos. We acknowledge the
assistance provided by Carla Chorley, Troy Moore, Kathi Shea, and Heather
Siefers in the establishment of a specimen and vaccine repository for our
trial and in their continued assistance with the handling and shipment of
specimens. We would like to acknowledge Gary Dubin, Anne Schuind,
Frank Struyf, Kelechi Lawrence, Darrick Fu, and Bruce Innis from GSK
Biologicals for their contribution to discussions regarding trial conduct
and Francis Dessy and Catherine Bougelet for HPV16/18 antibody testing.
We would like to thank members of the Data and Safety Monitoring Board
charged with protecting the safety and interest of participants in our trial
(Steve Self [Chair], Adriana Benavides, Luis Diego Calzada, Ruth Karron,
Ritu Nayar, and Nancy Roach) and members of the external Scientiﬁc HPV
Working Group who have contributed to the success of our efforts over the
years (Joanna Cain, Chair, Diane Davey, David DeMets, Francisco Fuster,
Ann Gershon, Elizabeth Holly, Silvia Lara, Henriette Raventós, Wasima
Rida, Luis Rosero-Bixby, Kristen Suthers, Sarah Thomas, and Raphael
Viscidi). We thank Nora Macklin for her support in preparing the article
Affiliations of authors: Infections and Immunoepidemiology Branch,
Division of Cancer Epidemiology and Genetics (ARK, AH), Hormonal and
Reproductive Epidemiology Branch, Division of Cancer Epidemiology and
Genetics (MES), Biostatistics Branch, Division of Cancer Epidemiology and
Genetics (SW), Clinical Genetics Branch, Division of Cancer Epidemiology
and Genetics (MS), Breast and Gynecologic Cancer Research Group,
Division of Cancer Prevention (DS), and Laboratory of Cellular Oncology,
Center for Cancer Research (JTS, DRL), National Cancer Institute, National
Institutes of Health, Rockville, MD; Proyecto Epidemiológico Guanacaste,
Fundación INCIENSA, San Jose, Costa Rica (ACR, CP, RH, PG, SJ); DDL
Diagnostic Laboratory, Voorburg, the Netherlands (WQ); Information
Management Systems, Rockville, MD (JS); Early Detection and Prevention
Section, International Agency for Research on Cancer, Lyon, France (RH).