Non-Inferiority Trial Comparing Immunogenicity From 1-Dose of Bivalent HPV Vaccine in Girls to 3-Doses of Quadrivalent Vaccine in Women: The PRIMAVERA-ESCUDDO Trial ("Puente de Respuesta Inmunológica Para Mejorar el Acceso a Vacunas y Erradicar el Cancer")
Phase: PHASE3
Conditions: Human Papillomavirus-Related Cervical Carcinoma
Sponsor: National Cancer Institute (NCI)

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Title: Non-inferiority trial comparing immunogenicity from 1-dose of bivalent HPV vaccine in 
girls to 3-doses of quadrivalent vaccine in women: The PRIMAVERA-ESCUDDO Trial (“Puente de 
Respuesta Inmunológica para Mejorar el Acceso a Vacunas y ERrAdicar el cancer”) 
Study Phase: Phase IIIb 
Study Vaccines: The FDA approved bivalent HPV16/18 virus-like particle with AS04-adjuvant 
vaccine (Cervarix) and the FDA approved HPV6/11/16/18 virus-like particles with Alum-adjuvant 
vaccine (Gardasil) 
Candidate regimen: One dose of Cervarix  
Control regimen: Three doses of Gardasil 
NCI Protocol number: 19-C-009 
NCI Principal Investigator: Aimée R. Kreimer PhD (NCI) on behalf of the PRIMAVERA-ESCUDDO 
Study team 
Costa Rica Principal Investigator: Bernal Cortes PharmDr (ACIB-FUNIN) on behalf of the 
PRIMAVERA-ESCUDDO Study team 
Study Sponsor: US National Cancer Institute 
 
Protocol Version/Date:  
Version 1.0: 30 October 2018 
Version 1.1: 28 November 2018 
Version 1.2: 13 December 2018 
Version 1.3: 13 March 2019 
Version 1.4: 20 June 2019 
Version 1.5: 15 June 2022 
Version 1.6: 28 February 2023 
 
ClinicalTrials.gov ID: NCT03728881 

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PRIMAVERA-ESCUDDO Study Team: 
US National Cancer Institute, National Institutes of Health 
Division of Cancer Epidemiology and Prevention 
Mitchell H. Gail 
Allan Hildesheim 
Aimée R. Kreimer 
Ligia Pinto 
Danping Liu 
 
Center for Cancer Research 
Doug Lowy 
John Schiller 
 
Agencia Costarricense de Investigaciones Biomédicas (ACIB) – Fundación INCIENSA 
Bernal Cortes 
Paula Gonzalez (deceased) 
Carolina Porras 
Byron Romero 
Ariane Castrillo 
 
International Agency for Research on Cancer 
Rolando Herrero 
Participating Institutions:  
US National Cancer Institute, National Institutes of Health, located at 9609 Medical Center 
Drive, Bethesda, MD 20892, USA. 
 
Agencia Costarricense de Investigaciones Biomédicas (ACIB) – Fundación INCIENSA, located at 
Solarium, frente Aeropuerto Daniel Oduber, bodega 8C, Liberia, Liberia, Guanacaste, 50101 
Costa Rica. 
 
International Agency for Research on Cancer, World Health Organization, located at 150 Cours 
Albert Thomas 69372 Lyon, France 
 
 

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LIST OF ABBREVIATIONS 
ACIB 
Agencia Costarricense de Investigaciones Biomédicas 
AS04 
Adjuvant system 04 (Monophosphoryl lipid A and aluminum phosphate) 
ATP 
According to protocol 
BCR 
B cell receptor 
°C 
Degrees Centigrade 
CDC 
U.S. Centers for Disease Control and Prevention 
CI 
Confidence interval 
CIN 
Cervical intraepithelial neoplasia 
CIN2/3 
Cervical intraepithelial neoplasia grades 2/3, adenocarcinoma in situ, and 
invasive cervical cancer 
PRIMAVERA 
Puente de Respuesta Inmunológica para Mejorar el Acceso a Vacunas y 
ERrAdicar el cancer (PRIMAVERA-ESCUDDO) 
CVT 
Costa Rica HPV Vaccine Trial  
DCEG 
Division of Cancer Epidemiology and Genetics 
DSMB 
Data and Safety Monitoring Board  
eCRF 
Electronic Case Report Form 
ELISA 
Enzyme-linked immunoabsorbent assay 
EMA 
European Medicines Agency 
ESCUDDO 
A scientific evaluation of one or two doses of vaccine against human 
papillomavirus: the ESCUDDO study (“Estudio de Comparacion de Una y 
Dos Dosis de Vacunas Contra el Virus de Papiloma Humano (VPH)”) 
ERP 
DCEG Ethics Review Panel 
FDA 
U.S. Food and Drug Administration 
FWA 
Federal-wide assurance 
GCP 
Good clinical practices 
GEE 
Generalized estimating equation 
GMT 
Geometric mean titer 
GSK 
GlaxoSmithKline 
HPV 
Human papillomavirus 
IARC 
International Agency for Research on Cancer 
ICD 
International Classification of Diseases 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IOM 
Institute of Medicine 
IRB 
Institutional Review Board 
ITT 
Intent to treat 
L1 
Papillomavirus structural proteins 
LLPC 
Long lived plasma cells 
LMIC 
Low to middle income country 
MD 
Maryland, USA or Doctor of Medicine 
mL 
Milliliter 

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MPL® 
Monophosphoryl lipid A (Corixa Corporation, Seattle, Washington, USA 
[Formerly Ribi]) 
NCI 
U.S. National Cancer Institute 
NIH 
U.S. National Institutes of Health 
OW 
Outreach worker 
PAHO 
Pan American Health Organization Technical Advisory Group 
PATRICIA 
PApilloma TRIal against Cancer In young Adults 
PID 
Participant study identification 
RBC 
Red blood cells 
RCT 
Randomized controlled trial 
SAE 
Serious adverse event 
SAG 
Scientific Advisory Group 
SAGE 
Strategic Advisory Group of Experts 
SAS 
Statistical Analysis System 
SEAP-NA 
Secreted alkaline phosphatase neutralization assay 
SOP 
Standard operating procedure 
TLR-4 
Toll-like receptor 4 
US or USA 
United States of America 
VE 
Vaccine Efficacy 
VAERS 
Vaccine Adverse Events Reporting System 
VLP 
Virus-like particle 
WHO 
World Health Organization 
 
 
 

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TABLE OF CONTENTS 
TABLE OF CONTENTS ..................................................................................................................................... 5 
SUMMARY ..................................................................................................................................................... 7 
Background and Rationale ............................................................................................................................ 9 
Evidence and biological rationale supporting protection by a single dose of the HPV vaccine ............. 11 
Rationale for the proposed Immunobridging Study ............................................................................... 12 
Antibody response to HPV vaccination is dependent on number of doses, time since last dose, vaccine 
product, and age and serostatus at vaccination ..................................................................................... 13 
Expectation of Non-inferiority in Immunobridging Study ...................................................................... 16 
STUDY OBJECTIVES ...................................................................................................................................... 17 
Primary Objective ................................................................................................................................... 18 
Secondary Objectives .............................................................................................................................. 19 
STUDY DESIGN AND METHODS OF TRIAL ................................................................................................... 19 
Overview ................................................................................................................................................. 19 
Selection of Study Vaccines .................................................................................................................... 21 
Selection of Gardasil (and not Gardasil9) as control vaccine product .................................................... 22 
Selection of HPV 16 and HPV 18 antibody levels as outcome ................................................................ 22 
Target populations .................................................................................................................................. 22 
Costa Rica as the study setting ............................................................................................................... 23 
Specimen collection ................................................................................................................................ 23 
Eligibility criteria ..................................................................................................................................... 23 
Inclusion criteria .................................................................................................................................. 23 
Exclusion criteria ................................................................................................................................. 24 
Deferral criteria at enrollment: ........................................................................................................... 24 
Contraindications to subsequent vaccination (Gardasil group) .......................................................... 24 
Concomitant Medications/Vaccinations ............................................................................................. 25 
Outreach and Enrollment Visits for Both Cervarix and Gardasil Groups (0 months) ............................. 25 
Gardasil Group Followup Vaccination Visits (2 and 6 months) ............................................................... 26 
Active Follow-up of Both Groups (Months 1, 12, 24, and 36) ................................................................ 27 
Home Visits During Follow-up................................................................................................................. 27 
Procedures and data collection .............................................................................................................. 27 
Medical history .................................................................................................................................... 27 

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Assessment of HPV vaccination outside the research study .................................................................. 28 
Urine Samples (Vaccination Visits only) .............................................................................................. 28 
Blood collection ................................................................................................................................... 28 
Vaccine Procedures ............................................................................................................................. 29 
Blood preparation, storage and shipment .............................................................................................. 29 
Expected Adverse Events and Adverse Event Monitoring and Reporting .............................................. 29 
Protocol Deviation Monitoring and Reporting ....................................................................................... 31 
Data collection and electronic records ................................................................................................... 31 
Serology .................................................................................................................................................. 32 
Efforts to maximize participation and retention .................................................................................... 33 
Approval of the exiting of participants from the study .......................................................................... 34 
ANALYSIS PLAN ........................................................................................................................................... 34 
Primary Analytical Cohort ....................................................................................................................... 34 
Immunogenicity Endpoint ....................................................................................................................... 35 
Analysis ................................................................................................................................................... 35 
Power and Sample Size Considerations .................................................................................................. 36 
HUMAN SUBJECTS PROTECTIONS ............................................................................................................... 37 
Institutional Approvals ............................................................................................................................ 37 
Justification for exclusions and dose schedule ....................................................................................... 37 
Safeguards for vulnerable populations ................................................................................................... 38 
Consent process and documentation ..................................................................................................... 40 
Subject privacy and confidentiality of data, including plans for sharing, future use, and disposition of 
study materials/biologic specimens ....................................................................................................... 42 
Plan for return of individual study results to participants ...................................................................... 42 
Evaluation of anticipated benefits and risks/discomforts ...................................................................... 43 
Compensation ......................................................................................................................................... 44 
ETHICAL CONSIDERATIONS AND TRIAL OVERSIGHT ................................................................................... 44 
Data and Safety Monitoring .................................................................................................................... 44 
Delivering trial results to participants and actions if study shows one dose failure. ............................. 44 
Working Group ........................................................................................................................................ 45 
Regulatory Body Review ......................................................................................................................... 45 

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DATA SHARING PLAN .................................................................................................................................. 45 
REFERENCES ................................................................................................................................................ 47 
NCI IRB/ERP AMENDMENTS AND APPROVAL DATES.................................................................................. 52 
 
SUMMARY 
Human papillomavirus (HPV) vaccination successfully prevents targeted HPV infections, related 
precancerous lesions, and, ultimately, cervical and other HPV-related cancers.  Despite the 
established efficacy of the HPV vaccine, most girls living in areas with the greatest risk for 
cervical cancer are not being vaccinated. The cost and logistical difficulties of vaccinating girls 
with the recommended multiple-dose administration has been a significant impediment to 
preventing this often-fatal cancer.   
The goal of this research is to provide convincing and actionable evidence for regulatory bodies 
that a single dose of the HPV vaccine will elicit an immune response sufficient to protect against 
targeted HPV infections and subsequent neoplasms. This study will provide earlier and 
complementary results to the definitive 24,000-girl study evaluating the vaccine efficacy for a 1-
dose regimen against virologic outcomes, which is underway in Costa Rica and is scheduled to 
be completed in 2024/5. That study, called ESCUDDO, is being conducted by the U.S. National 
Cancer Institute (NCI) and the Agencia Costarricense de Investigaciones Biomédicas (ACIB). 
Our specific objective is to show that the immune response for 1-dose of the bivalent vaccine 
Cervarix in girls 9-14 years old is non-inferior to the immune response for 3-doses of the 
quadrivalent vaccine Gardasil in women 18-25, a dosage/population combination with 
demonstrated efficacy. We emphasize that we are evaluating a single dose using the more 
immunogenic vaccine (i.e. Cervarix) in the population (i.e. 9-14-year-old girls) that has the 
stronger immune response. Therefore, despite a 1-dose regimen eliciting lower titers than a 3-
dose regimen when using the same vaccine and population, we hypothesize that 1-dose of 
Cervarix in girls will be non-inferior to 3-doses of Gardasil in women. We would expect that a 
successful demonstration of non-inferiority will provide a direct regulatory pathway for label 
change, which would in turn promote early adoption of and preparation for 1-dose vaccine 
programs by relevant agencies (i.e. WHO). We note that new vaccines or new administrations 
of a vaccine have, in the past, been recommended or approved for licensure by demonstrating 
that the immune response for the new vaccination plan is non-inferior to the immune response 
from an existing, approved vaccination plan. For the HPV vaccine, these immunobridging 
studies have informed the recommendation for a two-dose administration, and vaccination in 
9-14 year old girls and boys. Scientifically, this study can provide additional evidence that 

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subunit vaccines with repetitive structures elicit sufficient antibody responses to confer 
protection and will demonstrate the importance of the adjuvant system to maximize plateau 
titer levels. 
From this effort, we anticipate one of two outcomes, which are summarized below along with 
their implications.  
Outcome #1:  The HPV-16 and HPV-18 antibody levels at 24 and/or 36 months from 1-dose of 
Cervarix in girls 9-14 years old are non-inferior to 3-doses of Gardasil in women 18-25 years 
old. The implication from this result is that 1-dose of Cervarix will be efficacious for protecting 
against incident HPV 16/18 infection and associated precancerous lesions. The result could lead 
to provisional approvals supporting a 1-dose regimen of the bivalent HPV vaccine by 
international and regulatory agencies (e.g., WHO, FDA, and EMA), contingent on the definitive 
corroboration of efficacy from ESCUDDO, which will deliver its results three to four years later.  
 
Outcome #2:  The HPV-16 and/or HPV-18 antibody levels at 24 and 36 months from 1-dose of 
Cervarix in girls 9-14 years old cannot be demonstrated to be non-inferior to 3-doses of 
Gardasil in women 18-25 years old. The implication from this result would be that the vaccine 
efficacy of a single dose will need to be directly evaluated by demonstration of protection 
against pertinent virologic outcomes, as planned in our ongoing 1-dose ESCUDDO study. 
 
 

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Background and Rationale 
Persistent infection with carcinogenic genotypes of the human papillomavirus (HPV) is a 
necessary causal event in cervical carcinogenesis (1, 2). HPV exposure typically begins when 
individuals become sexually active, reaching peak incidence during the third decade of life (3). 
More than one half of carcinogenic HPV infections clear spontaneously within a year and 
approximately 90% resolve within several years (4, 5); however, infections that persist may 
progress to cancer precursors, and then to invasive cancer, typically after more than a decade 
(5). Worldwide, there are approximately 530,000 cervical cancers diagnosed annually and 
265,000 deaths, 88% of which occur in low income nations, where cervical cancer is the leading 
cause of cancer mortality among women (6). Approximately 50% of cervical cancer is caused by 
HPV16, an additional 20% by HPV18, and the remaining 30% by 10 other carcinogenic types 
collectively (7). Accordingly, preventing carcinogenic HPV infections, especially HPV16 and 18, 
would dramatically reduce cervical cancer incidence and mortality. In addition, reducing HPV16 
infections would eliminate a substantial proportion of vulvar, vaginal, anal, penile, and 
oropharyngeal cancers (1, 2).  
There are currently three available HPV vaccines: Cervarix® (GlaxoSmithKline, Rixensart, 
Belgium) directed against HPV16 and 18, Gardasil® (Merck & Co., Whitehouse Station, NJ) 
directed against HPV16, HPV18 and the two HPV types causing genital warts, HPV6 and 11, and 
Gardasil-9® (Merck & Co., Whitehouse Station, NJ) directed against HPV 16, 18, 6, 11, 31, 33, 
45, 52, and 58. Three doses of the commercially available HPV vaccines were demonstrated to 
be highly efficacious in preventing targeted carcinogenic HPV infections and related cervical 
cancer precursors and cancers. The vaccines also protect against infection at other sites at 
which HPV-related cancers develop (8-10). The HPV vaccines contain non-infectious, non-
carcinogenic, virus-like particles (VLPs) consisting of L1 protein, the primary structural 
component of the HPV capsid. In addition to differences in the HPV types covered, Merck 
vaccines employ an aluminum hydroxyphosphate sulfate adjuvant, whereas the Cervarix 
adjuvant is AS04 (monophosphoryl lipid A and aluminum phosphate), which acts as a toll-like 
receptor-4 agonist that stimulates innate and adaptive immune responses and yields a 
significantly higher antibody response compared to the quadrivalent HPV vaccine (11).  
The bivalent and quadrivalent HPV vaccines have demonstrated high efficacy against cervical 
intraepithelial neoplasia (CIN) grades 2/3 (CIN2/3) (cervical precancer) attributable to HPV16 
and HPV18 when administered as a three-dose series in randomized, double-blind, multi-
centric phase III trials conducted among women ages 15-26 years (12-14). Based on 
immunologic non-inferiority, regulatory agencies have approved and recommended a two-dose 
schedule of these vaccines (15-18). Specifically, the FDA, EMA, and WHO approvals and 

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recommendations for two-dose administrations came between December 2013 and October 
2016 (15-19).  
Analyses of population-level data accumulated in the first five years following implementation 
of an organized nationwide vaccination program with the quadrivalent HPV vaccine in Australia 
indicate that rates of condyloma and CIN have rapidly declined, providing an early indicator of 
vaccine effectiveness, together with evidence of herd immunity (20-23). Similarly, population-
level data from Scotland accumulated in the first seven years following implementation of an 
organized nationwide vaccination program with the bivalent HPV vaccine show prevalence of 
HPV vaccine type and non-vaccine type, as well as CIN2+, are dropping precipitously in both 
vaccinated and unvaccinated women (i.e.: demonstration of herd immunity) (24).  
Consequently, the WHO states that the currently-available HPV vaccines provide similar 
protection against cervical cancer, and all girls in the recommended age range should be 
vaccinated with two doses of any of the HPV vaccines (19).  Further, the WHO and the CDC 
provide ongoing monitoring of the HPV vaccines for safety-related outcomes: they report that 
HPV vaccines have similar safety profiles to other licensed vaccines (25, 26). 
HPV vaccination of children prior to sexual initiation has been implemented progressively in 
high-income countries (and now in many middle-income countries, particularly in Latin 
America) since the vaccines were first licensed in 2006. More recently, a few low-income 
countries, such as Bhutan and Rwanda, adopted similar vaccination strategies for pre-
adolescents. Yet, while HPV vaccines are currently licensed for use in over 100 countries, active 
vaccination programs are limited to about 50 countries, most of which are high-income 
countries (27). Costs and logistical challenges pose barriers to administration of a complete 
three-dose, or even a two-dose series, especially in countries with limited resources but high 
cervical cancer burden. In Costa Rica, the three HPV vaccines have been licensed by the 
Ministry of Health and, following support by the National Immunization Commission, were 
included in the National Immunization Program (under the Social Security System) starting in 
2019. They implemented a school-based approach that administers two doses of the 
quadrivalent HPV vaccine to girls in fourth grade (typically age 10; one birth cohort per calendar 
year). 
Scope of the Problem. Globally, fewer than 3% of age-eligible girls in low-income countries 
receive even a single dose of the HPV vaccines (27). Projecting current cervical cancer incidence 
and mortality trends in the face of low vaccine uptake, deaths from cervical cancer will increase 
dramatically in low-and middle-income countries over the next several years (28).  Even in some 
wealthier nations, completion of even a two-dose vaccine series continues to be a problem.  
Thus, there is an urgent need to expedite the evaluation of a single dose of the prophylactic 

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HPV vaccine, to present the resulting evidence to regulatory and/or recommending bodies, and 
ultimately to ensure that more girls receive the vaccine. 
Evidence and biological rationale supporting protection by a single dose of the HPV vaccine 
The first line of evidence to support that HPV vaccines will induce long term protection after a 
single dose is the finding that the vaccines consistently induce strong and durable neutralizing 
antibody responses against the targeted types among individuals who received only a single 
dose. For Cervarix, the GMT antibody titers after four years were only four-fold lower in one 
dose compared to three dose recipients (29), and this ratio has been maintained out to seven 
years (30). For Gardasil, a similar four-fold difference between the antibody levels in the one 
and three dose recipients were reported in a four-year interim analysis (31).  Note that we 
focus on the plateau levels of VLP antibodies and not peak titers immediately after the booster 
dose because the vast majority of exposures to sexually transmitted HPV infections will occur 
when vaccinated females are in the plateau phase. Further, in assessments of single- and multi-
dose regimens of the HPV vaccines, peak titers do not predict plateau titers. Presently, direct 
comparison between the levels induced by different vaccine products among one-dose 
recipients is not possible because of the differences in study design, populations, and serologic 
assays used. 
The second line of evidence that supports the hypothesis that a single dose of the HPV 
vaccine(s) may be sufficient is from post-hoc analyses of non-randomized groups from RCTS.  In 
these studies, the observed vaccine efficacy (VE) against incident, persistent HPV 16/18 
infections appears to be independent of the number of doses. For Cervarix, after four years of 
the follow-up in the Costa Rica Vaccine Trial, VE against incident infection by HPV16/18 for 
three doses was estimated to be 87% (95% CI=77 to 89%), while the VE for one dose was non-
significantly different (VE 100%; 95% CI=79-100%) (29). Similarly, high VE in both arms was 
observed at 7 years (30) and in the PATRICIA study (29). For Gardasil, after 7 years of follow-up 
in the India HPV vaccine trial, the frequencies of cumulative incident HPV 16 and 18 infections 
were similar and uniformly low regardless of the number of doses; the frequencies of HPV 16 
and 18 infections among the 1,180 women who received three doses was 0.9% and among the 
1,823 women who received one dose was 1.6%, compared to a frequency of 6.2% in 1,481 
unvaccinated women (32). 
The exceptional immunogenicity of HPV vaccines can largely be attributed to the structure of 
the HPV vaccine antigen. Typical subunit vaccines are composed of monomeric or low valency 
multimeric proteins or carbohydrate/protein conjugates.  In contrast, HPV VLPs are composed 
of 360 ordered protein subunits that form a particulate 55nm structure displaying a repetitive 
array of epitopes on their surface.  The engagement of these repetitive elements by the B cell 
receptors (BCRs) on naïve B cells is believed to transmit exceptionally strong activation and 

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survival signals to the cells leading to consistent induction of memory B cells, and, most 
importantly, long lived plasma cells (LLPCs) that continuously produce antibodies for many 
years (33).  Epitope spacing of 50-100Å appears to be critical.  This spacing is commonly found 
on microbial surfaces but not on body surfaces normally exposed to the systemic immune 
system. The particulate and repetitive structure of VLPs likely contributes to B cell immunity in 
several additional ways.  Particles of this size efficiently traffic to lymph nodes and are also 
efficiently phagocytized by antigen presenting cells for the initiation of immune responses and 
the generation of cognate T helper cells.  The poly-valency of VLPs also leads to stable binding 
of nature low avidity IgM and complement, promoting their acquisition by follicular dendritic 
cells, which are key components in inducing humoral immune responses in the lymph node.  
The hepatitis B vaccine is also considered a VLP but appears to be much less immunogenic after 
a single dose, perhaps because it has a fewer subunits and/or because they float in a lipid 
bilayer.  To summarize, by mimicking the key structural features of authentic viruses, the HPV 
VLPs consistently induce potent and long lasting humoral responses that more closely resemble 
the anti-virion responses to an acute virus infection or a single dose of a live-attenuated virus 
vaccine than the response to simple subunit vaccines (34).  
Rationale for the proposed Immunobridging Study 
There are pressing reasons to demonstrate that a single dose of the HPV vaccine is efficacious. 
These reasons led to the NCI’s large-scale trial entitled “A scientific evaluation of one or two 
doses of vaccine against human papillomavirus: the ESCUDDO study” (clinicaltrials.gov 
identifier: NCT03180034). However, the results from this definitive study will not be available 
until 2024/2025. There would be significant benefits to provide prospective evidence with a 
controlled trial prior to 2024 that 1-dose should be efficacious. Furthermore, this proposed 
study offers a complementary approach, through immunobridging, to obtaining a one-dose 
HPV vaccine indication, if warranted by the data.  
Indeed, the FDA and EMA have approved multiple changes to the vaccine label based on 
immunologic non-inferiority assessments in immunobridging studies (35). Specifically, the 
regulatory agencies (i.e. FDA (15, 36, 37) and EMA (8, 15-17, 38)) approved, and the 
international agencies (i.e. CDC (26) and WHO (19)) recommended, two-doses of the 
vaccination for boys and girls between the ages of 9 to 14. These changes to the vaccine label—
for two-doses (39, 40), a younger age (41-45), and boys (45, 46) — were based on studies 
demonstrating that the antibody levels, as measured by ELISA, at one month following the last 
vaccination in the new population or new administration were non-inferior to levels among 
women 15-25 years old, the ‘efficacy population’, at one month following the third vaccination 
(47, 48). 

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The proposed study will evaluate a single dose of a highly immunogenic vaccine (i.e. Cervarix) in 
a population (i.e. 9-14-year-old girls) that has the stronger immune response and compare that 
to the antibody levels in a population with demonstrated efficacy.  We now explain how, 
despite a 1-dose regimen eliciting lower titers than a 3-dose regimen when using the same 
vaccine and population, we hypothesize that 1-dose of Cervarix in girls will be non-inferior to 3-
doses of Gardasil in women. 
Antibody response to HPV vaccination is dependent on number of doses, time since last dose, 
vaccine product, and age and serostatus at vaccination 
HPV 16/18 antibody levels induced by the HPV vaccines depend on the number of doses, the 
time since the vaccine was administered, the vaccine product, and the age and serostatus at 
initial HPV vaccination. We first discuss the relationship between antibody levels and each of 
these characteristics. We then show that, based on these relationships, it is reasonable to 
expect that antibody levels at 36 months after vaccination should be similar in 9 to 14 year old 
girls receiving a single dose of Cervarix and 18 to 25 year old women receiving three doses of 
Gardasil. This conclusion is the cornerstone of our proposed study.  
Time since last dose: Among the women who receive three doses of Gardasil, the antibody 
levels peak immediately following the third vaccination and then decline to a more stable 
plateau. The boosting of the primary response generates a transient burst of antibodies, 
presumably due to the induction of short-lived plasma blasts from memory B cells, which 
transiently exaggerates the contribution of the booster doses to antibody titers.  
Einstein and colleagues (49) show that, among approximately 100 women 18-26 year old who 
received three doses of Gardasil, the GMTs for HPV 16 antibodies decrease from 10053 at 7 
months after the initial vaccination to 3265, 1183, 894, 653, and 750 at 12, 18, 24, 36, and 48 
months after vaccination. Similarly, the GMTs for HPV 18 antibodies decrease from 2258 at 7 
months to 596, 249, 175, 128, and 139 at 12, 18, 24, 36, and 48 months after vaccination.  
We emphasize that no similar peak is observed after the first dose of Cervarix (Figure 1). To 
illustrate this, in the Costa Rica HPV Vaccine Trial (CVT) among women who received three 
doses, the HPV16 GMT one-month post dose one of Cervarix was 598; this increased after the 
second and third doses to 2035 at 12 months, and then decreased to 1115, 899, and 748 at 24, 
36, and 48 months.  For HPV18, the 1-month GMT was 224, which increased following the 
second and third dose to 826 at 12 months, and then decreased to 470, 369, and 334 at similar 
time points (n.b. antibody titers cannot be compared between studies due to profound 
differences in immunologic assays). In contrast, among women who received only a single dose, 
the GMTs for HPV 16 antibodies at 12 months after the first vaccination was 114 and remained 
stable at 124, 135, and 137 at 24, 36, and 48 months (Figure 1; Table 1). Among these same 

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women, the GMTs for HPV 18 antibodies at 12, 24, 36, and 48 months after vaccination was 70, 
69, 73, and 70 (50). Given the association with time since vaccination and antibody titer, our 
evaluation is focused on antibody levels in the plateau phase (at least 24 or 36 months after 
vaccination), and not the peak titers, as we believe these are more relevant and predictive of 
long-term vaccine protection. 
Number of Doses: The immune response to HPV vaccination increases with the number of 
doses. At 36 months after the initial vaccination in CVT, the GMTs for HPV-16 and HPV-18 
antibody levels were respectively 6.6x (95% CI = 4.8-9.0) and 4.8x (3.5-6.4) higher among 
women who received 3-doses of Cervarix as compared to 1-doses (50). At 24 months in CVT, 
the GMTs for HPV-16 and HPV 18 were 9.1x (6.5-12.5) and 6.9x (5.0-9.4) higher, respectively, in 
3-dose Cervarix recipients (50). 
Vaccine Product: Cervarix elicits a higher immune response because of the AS04 adjuvant in its 
formulation. At 36 months after the initial vaccination, Einstein and colleagues demonstrated 
that the GMTs for HPV-16 and HPV-18 were 5.9x (3.9-9.4) and 12.5x (8.0-19.4) higher among 
women receiving 3-doses of Cervarix as compared to 3-doses of Gardasil (49). At 24 months 
after the initial vaccination, the GMTs for HPV-16 and HPV-18 were 5.8x (4.0-8.5) and 9.4x (6-
14) higher among women receiving 3-doses of Cervarix as compared to 3-doses of Gardasil (49). 
Although less relevant for our purposes because they compared Cervarix and Gardasil within 
three to six months after the third vaccination, two other studies demonstrated GMTs for HPV-
16 and HPV-18 were 3.3 to 6.6x higher among women who received Cervarix (51, 52). 
Age: The VLP antibody response tends to be higher at younger ages. Unfortunately, there are 
no available data that directly compares HPV antibody levels at 36 months after the single and 
only dose of Cervarix between 9-14 year old girls and 18-25 year old women. Instead, we 
discuss results from related studies evaluating the general relationship between age and 
immune response.  
At 36-months post-dose 1 among 3-dose recipients, the GSK trial HPV-012 found that GMTs for 
antibodies elicited by Cervarix to HPV-16 and HPV-18 were, respectively, 2.4x (1.6-3.5) and 1.9x 
(1.3-2.8) higher in girls 10-14 years old as compared to girls/women 15-25 years old (53). 
Similarly, Einstein and colleagues (49) demonstrated that GMTs for HPV 16 and HPV 18 
antibodies were 2.0x (1.3-3.1) and 1.7x (1.1-2.6) higher among 18-26 year olds as compared to 
27-35 year olds, again at 36 months among 3-dose recipients of Cervarix, with similar findings 
for Gardasil (Table 1).  
At peak levels one-month after the third dose of Gardasil, Block et al (43) demonstrated that 
GMTs for HPV-16 and HPV-18 were, respectively, 1.8x (1.5-2.2) and 2.0x (1.7-2.4) higher in girls 
10-15 years old as compared with girls/women 16-23 years old.  

15 
 
When focusing on girls within the 9-14 year old range, there is no evidence to suggest that 
antibody levels dramatically increase at menarche. Reisenger and colleagues (54) reported that 
GMTs for HPV-16 and HPV-18 were 1.5x and 1.6x higher in girls 9-12 years old as compared to 
girls 13-15 years old at 1 month after last vaccination among 3-dose recipients of Gardasil. 
Others (unpublished data, personal communication) have similarly found that the immune 
response is similar pre/post-menarche.  
HPV serostatus at time of initial vaccination: Finally, HPV serostatus at the time of HPV 
vaccination (i.e.: due to natural infection) does impact vaccine-induced plateau level. Petersen 
et al (55) reported that, among women who received three doses of Gardasil-9 in the per 
protocol population, HPV16 antibody titers 36-months after initial vaccination were 3.2x lower 
compared to HPV16 seropositive/HPV16 DNA negative women.  This latter group comprised 
~12% of the total study population and were excluded in the evaluations for licensure of the 
Merck HPV vaccines.  
Figure 1. HPV 16 and 18 GMTs by months and number of doses of Cervarix (adapted from ref 
(50)) 
 
 
 
10
100
1000
10000
0
12
24
36
48
HPV 16 and 18 GMTs by months and number of 
doses
1 dose  HPV 16
1 dose HPV 18
3 dose HPV 16
3 dose HPV 18

16 
 
Table 1. GMT ratios of the antibody response to HPV vaccination by number of doses administered, 
the time since last dose, the vaccine product, and age and serostatus at first vaccination. 
HPV 
type 
 
# of 
doses 
Comparison groups 
# of months post vaccination 
 
7 
24 
36 
Vaccines (Source) 
Ratio (95% CI) 
Ratio (95% CI) 
Ratio (95% CI) 
Studies comparing GMTs between number of vaccine doses 
HPV-16 
Cervarix † 
1, 3 
3 doses vs. 1 
9.1 (6.5-12.5) † 
9.1 (6.5-12.5) †  6.6 (4.8-9.0) † 
HPV-18 
Cervarix † 
1, 3 
3 doses vs. 1 
 
6.9 (5.0-9.4) † 
4.8 (3.5-6.4) † 
Studies comparing GMTs by vaccine product 
HPV-16 
Cervarix & Gardasil (49) 
3 
Cervarix vs. Gardasil 
 
5.8 (4.0-8.5) 
5.9 (3.9-9.4) 
 
Cervarix & Gardasil (52) 
3 
Cervarix vs. Gardasil 
4.3 ‡** 
 
 
HPV-18 
Cervarix & Gardasil (49) 
3 
Cervarix vs. Gardasil 
 
9.4 (6-14) 
12.5 (8.0-19.4) 
 
Cervarix & Gardasil (52) 
3 
Cervarix vs. Gardasil 
6.6 ‡** 
 
 
Studies comparing GMTs across age groups 
HPV-16 
Gardasil (43) 
3 
10-15 vs. 16-23 y/o 
1.8 (1.5-2.2)  
 
 
 
Cervarix (53) 
3 
10-14 vs. 15-25 y/o 
2.4 (1.6-3.5) 
 
 
 
Cervarix (49) 
3 
18-26 vs. 27-35 y/o 
 
 
2.0 (1.3-3.1) 
 
Gardasil (54) 
3 
9-12 vs. 13-15 y/o 
1.5 †† 
 
 
HPV-18 
Gardasil (43) 
3 
10-15 vs. 16-23 y/o 
2.0 (1.7-2.4)  
 
 
 
Cervarix (53) 
3 
10-14 vs. 15-25 y/o 
1.9 (1.3-2.8) 
 
 
 
Cervarix (49) 
3 
18-26 vs. 27-35 y/o 
 
 
1.7 (1.1-2.6) 
 
Gardasil (54) 
3 
9-12 vs. 13-15 y/o 
1.6 †† 
 
 
Seropositivity at time of vaccination  
HPV-16 
Cervarix  
1 
SP vs SN 
 
3.8 
3.8 
 
Cervarix  
3 
SP vs SN 
 
1.5 
1.5 
 
Gardasil-9 (55) 
1 
SP vs SN 
 
– 
– 
 
Gardasil-9 (55) 
3 
SP vs SN 
 
3.2 
3.2 
HPV-18 
Cervarix  
1 
SP vs SN 
 
2.0 
2.0 
 
Cervarix 
3 
SP vs SN 
 
1.7 
1.7 
 
Gardasil-9 (55) 
1 
SP vs SN 
 
– 
– 
 
Gardasil-9 (55) 
3 
SP vs SN 
 
5.0 
5.0 
GMT, Geometric mean titers; CI, Confidence interval; y/o, years old, SP seropositive; SN seronegative 
† Costa Rica Vaccine Trial, unpublished results 
‡ 1-to-6 months after the 3rd vaccine dose 
†† 1-month after the 3rd vaccine dose 
** Estimated from data provided in the paper, reported p-values for Cervarix vs. Gardasil = 0.0001 
Expectation of Non-inferiority in Immunobridging Study 
Based on the acceptance of immunogenicity as a surrogate for vaccine efficacy, our objective is 
to demonstrate that the immune response for 1-dose of Cervarix in girls 9-14 years old is non-
inferior to the immune response for 3-doses of Gardasil in women 18-25 years old, a 
dosage/population combination with demonstrated efficacy. Although there are no direct 

17 
 
comparisons of the immune response in these two groups, we can extrapolate based on the 
following equation: 
GMT(1-D, C, 9-14yo)      GMT(1-D, C, 18-25yo)     GMT(3-D, C, 18-25yo)     GMT(1-D, C,9-14yo) 
----------------------------- = ------------------------------ x ------------------------------ x ---------------------------- 
GMT(3-D, G, 18-25yo)      GMT(3-D, C, 18-25yo)     GMT(3-D, G,18-25yo)     GMT(1-D, C,18-25yo) 
 
In terms of notation, 1-D and 3-D abbreviate 1-dose and 3-dose administrations and C and G 
abbreviate Cervarix and Gardasil. On the right side, we have three ratios that were discussed in 
the section above (we focus on HPV16, as the predicted ratio is higher for HPV18).  
At 24-months, the time of the proposed interim analysis, the 1-Dose to 3-Dose ratio should be 
greater than 1/9.1=0.11; the Cervarix to Gardasil ratio should be greater than 5.8; the 9-14-
year-old to 18-25-year-old, with some assumptions should be greater than 1.8 (see numbers in 
bold above). Taking this evidence together, we predict 
GMT(1-D, C, 9-14yo)             
------------------------------- > 0.11 x 5.8 x 1.8 = 1.14 
GMT(3-D, G, 18-25yo)           
 
At 36-months, the time of the proposed analysis, the 1-Dose to 3-Dose ratio should be greater 
than 1/6.6=0.15; the Cervarix to Gardasil ratio should be greater than 5.9; the 9-14-year-old to 
18-25-year-old, with some assumptions should be greater than 2.0. Taking this evidence 
together, we predict 
GMT(1-D, C, 9-14yo)             
------------------------------- > 0.15 x 5.9 x 2.0 = 1.77 
GMT(3-D, G, 18-25yo)           
 
Therefore, based on the available evidence, we believe that the plateau antibody GMT for 1-
dose of Cervarix in girls 9-14 years old is non-inferior to the plateau antibody GMT for 3-doses 
of Gardasil in women 18-25 years old.  Indeed, the above arithmetic suggests the 1-dose 
Cervarix regimen in adolescents may indeed elicit an even higher antibody response compared 
to the 3-dose regimen of Gardasil in women.  
STUDY OBJECTIVES 
The objective of this study is to demonstrate that the immune response for 1-dose of Cervarix 
in girls 9-14 years old is non-inferior to the immune response for 3-doses of Gardasil in women 
18-25, a dosage/population combination with demonstrated efficacy. As immunobridging 

18 
 
studies are an accepted approach for approving new vaccine schedules and indications, we 
believe that this study, if it successfully demonstrates non-inferiority, will provide a basis for 
label change by regulatory bodies and inform recommendations by international agencies (i.e., 
WHO; immunobridging with readout at the time of plateau is addressed by the TRS962). This 
evidence can arrive 2-3 years prior to the completion of the ESCUDDO study (NCI 17-C-N108, 
CEC-ICIC-E093-2017).  
We hope to provide regulators and public health policy makers with the necessary scientific 
information required to make evidence-based decisions related to large-scale implementation 
of sustainable, cost-effective HPV vaccine programs. Specifically, evidence of strong protection 
from a single-dose of Cervarix may enable nations to achieve extensive population coverage by 
vaccinating a much larger number of HPV-naïve individuals, which, in combination with herd 
immunity, will result in decreased cervical cancer incidence and mortality over time, in addition 
to reductions in other cancers. It would also support implementation policies that prioritize 
wider population coverage with one dose over initiating and completing a two-dose protocol 
region-by-region, which inevitably risks delays and gaps in coverage in some areas.  
Primary Objective 
To demonstrate that the immunogenicity (as determined by ELISA) of a single dose of Cervarix 
in 9-14 year old girls is non-inferior to the immunogenicity of three doses of Gardasil, 
administered at 0, 2, and 6 months, in 18-25 year old women 36 months after initial 
vaccination, with an interim analysis at 24 months after initial vaccination. 
Either of following criteria will qualify as non-inferiority: 
- 
The interim analysis will assess non-inferiority at 24 months. Non-inferiority will be 
demonstrated if the lower limits for the two-sided 99% confidence intervals of the GMT 
ratios (1-Dose Cervarix divided by 3-Doses of Gardasil at 24 months) for both HPV-16 and 
HPV-18 antibody levels exceed 0.67.  
– The primary analysis will assess non-inferiority at 36 months.  Non-inferiority will be 
demonstrated if the lower limits for the two-sided 96% confidence intervals of the GMT 
ratios (1-Dose Cervarix divided by 3-Doses of Gardasil at 36 months) for both HPV-16 and 
HPV-18 antibody levels exceed 0.67.  
Endpoint definition for immunogenicity: We will measure HPV-specific serum antibodies using 
the VLP-based direct enzyme-linked immunoassay (ELISA) on serum collected at 24 and 36 
months after initial vaccination. The 36-month timepoint is the primary objective; an interim 
analysis will be conducted at 24-month to determine if non-inferiority can be achieved earlier, 
with appropriate penalty to the α-level. In addition to the ELISA, other assays may be utilized to 

19 
 
better understand the response to vaccination, such as the measurement of neutralizing 
antibodies and avidity.  
Cohort for assessment of immunogenicity:  Participants who receive the appropriate number of 
doses during the predefined vaccination windows and do not have serologic evidence for the 
corresponding HPV type considered in the analysis (i.e., are HPV16 seronegative for HPV16 
analysis, and are HPV18 seronegative for HPV18 analysis) at baseline will serve as the analytical 
cohort for evaluation of the study objective.  
Secondary Objectives 
1) To compare the distributions of both HPV-16 and HPV-18 antibodies levels, assessed at 24 
and 36 months after initial vaccination, following a single dose of Cervarix in 9-14 year old 
girls with the corresponding distributions  following three doses of Gardasil, administered at 
0, 2, and 6 months, in 18-25 year old women. 
2) To compare proportions with seroconversion based on both HPV-16 and HPV-18 antibody 
levels, assessed at 24 and 36 months after initial vaccination, following a single dose of 
Cervarix in 9-14 year old girls and following three doses of Gardasil, administered at 0, 2, 
and 6 months, in 18-25 year old women. 
3) To compare GMTs, distributions, and seroconversion proportions for HPV-16 and HPV-18 
antibodies, assessed at 24 and 36 months after initial vaccination, following a single dose of 
Cervarix in 11-14 year old girls and three doses of Gardasil, administered at 0, 2, and 6 
months, in 18-25 year old women. 
4) To compare GMTs, distributions, and seroconversion proportions for HPV-16 and HPV-18 
antibodies, assessed at 1-month after vaccination and 1-year after vaccination, following a 
single dose of Cervarix in 9-10 year old girls and following a single dose of Cervarix in 11-14 
year old girls.  
5) To evaluate whether baseline variables (e.g., age, enrollment date, and district) are 
associated with GMTs, distributions, and seroconversion proportions for HPV-16 and HPV-
18 antibodies, assessed at 24 and 36 months after initial vaccination. 
We note that Secondary Objectives 3 and 4 are, in part, included in this study because of the 
introduction of Costa Rica’s national HPV vaccination program in 2019, which aimed to 
vaccinate 10-year-old girls. 
STUDY DESIGN AND METHODS OF TRIAL 
Overview 
The PRIMAVERA study is a non-randomized, open-label, Phase IIIb clinical trial comparing 1 
dose of the bivalent HPV vaccine (Cervarix) in girls to 3 doses of the quadrivalent HPV vaccine 

20 
 
(Gardasil) in women. We will work within selected regions in Costa Rica and invite girls 9-14 
years old (N=between 520 and 620; N=between 173 and 206 9-10 years and N=between 347 
and 414 11-14 years) and women 18-25 years old (N=between 520 and 620) to visit dedicated 
study clinics. Randomization will not be utilized in this trial because age indicates vaccine 
product and number of doses. Therefore, vaccine product blinding is not necessary. Girls will be 
invited with their parents/legal guardians. At the first (baseline) study visit, the study will be 
described in detail. Written informed consent will be obtained from adult participants and 
parents/legal guardians of minor participants. Written assent will be obtained from girls 12-14 
years old; girls younger than 12 will be provided with a study information document and their 
verbal agreement to participate will be recorded in the study management system.  
The potential participant’s medical history will be assessed by a clinician, including previous 
HPV vaccination. If needed for eligibility determination and at the clinician’s discretion, the 
clinician can perform a physical examination to confirm good general health. If the potential 
participant is deemed potentially eligible, she will undergo blood collection. Participants 12 and 
older will be required to have a negative urine-based pregnancy test prior to vaccination, and 
sexually active participants will be instructed to use effective birth control methods (including 
abstinence) for 60 days after the last vaccination. 
The initial vaccination will be administered at this baseline study visit; this will be either the first 
dose of Gardasil or the single dose of Cervarix. Participants will be monitored for adverse 
events at the clinic for 15 minutes following vaccination. Appropriate medical treatment will be 
readily available in the rare event that an anaphylactic reaction develops. A real-time contact 
service (such as a toll-free number or text messaging system) will be staffed for the duration of 
the trial so that participants may contact the trial’s clinical team in case of study-related 
medical concerns or questions. 
Among women scheduled to receive Gardasil, the second and third vaccine visit will occur two 
and six months following the initial baseline visit. Qualified staff and clinicians will conduct 
these study visits. After a negative urine-based pregnancy test, the visit will conclude with 
vaccination and monitoring for adverse events. All adverse events, serious and not serious, will 
be documented.  
All participants will have blood collected at the 12, 24, and 36 month follow-up visits, either in a 
clinic or home setting (Table 2). For participants in the Cervarix group, blood also will be 
collected at a follow-up visit one month after vaccination. Each girl is expected to have five 
study visits, one for vaccination and four follow-up visits. Each woman is expected to have six 
study visits, three for vaccination and three for follow-up. 
Table 2. Proposed study visits for the two groups.  

21 
 
  
Visit type (months) 
Age 
V00* 
V01 
V02* 
V06* 
V12 
V24 
V36 
9-14 years 
X 
X 
 
 
X 
X 
X 
18-25 years 
X 
 
X 
X 
X 
X 
X 
*Indicates vaccination visit 
Throughout the duration of the trial, we will monitor plans for country-wide implementation of 
HPV vaccination, which will target fourth-grade girls (usually 10 year olds). At the time when 
the country begins vaccination, we will initiate procedures for ceasing enrollment of 9- and 10-
year olds according to the SOP requested by the DSMB. Note, our recruitment plan for the 
younger cohort ensures that we will include 347 girls that will not be targeted by the national 
vaccination program.  
Finally, if at 36-months the GMT for 1 dose at the group level is inferior to that of 3 doses, all 1-
dose girls will be invited to receive the 2nd dose of Cervarix. 
Selection of Study Vaccines 
Both vaccines to be used in this study are licensed in the US and Costa Rica. As described, the 
bivalent HPV vaccine is produced by GSK (Cervarix®) and contains HPV 16 and 18 antigens, the 
quadrivalent vaccines is produced by Merck (Gardasil®) and additionally contain HPV 6 and 11 
antigens. Besides the differences in HPV types included, the bivalent vaccine has a proprietary 
adjuvant called AS04, consisting of monophosphoryl lipid A (MPL) and aluminum hydroxide. 
MPL is a detoxified bacterial lipopolysaccharide which is a TLR-4 agonist involved in activation 
of innate and adaptive immune responses. The quadrivalent vaccine has aluminum 
hydrophosphate sulfate as adjuvant.  
The quadrivalent HPV vaccine is now included in the Costa Rica national vaccine program as a 
two-dose administration for 10-year old girls, which aligns with the official recommendation of 
PAHO, which recommended a schedule of two doses for adolescents under the age of 15 years 
in 2013 (50,51). Beyond the PAHO recommendation, the Cervarix product details (Version 
number: GDS024/IPI019) state that the two-dose regimen has been approved in Costa Rica for 
girls younger than 15 ("9-14 years inclusive: Two doses of 0.5 ml each. The second dose given 
between 5 and 13 months after the first dose or three doses of 0.5 ml each at 0, 1 and 6 
months”). The Gardasil product details (quadrivalent vaccine, MSD 70031548/00-1) state that 
two doses of 0.5 ml each can be given to girls between 9 to 13 years at 0 and 6 months or 0 and 
12 months. Per this same document, for all others, three doses of 0.5 ml each should be given 
at 0, 2 and 6 months. 

22 
 
Selection of Gardasil (and not Gardasil9) as control vaccine product 
Efficacy studies using the quadrivalent HPV vaccine were conducted on women; for HPV types 
16 and 18, the nonavalent HPV vaccine was subsequently approved based on observed non-
inferior immunogenicity among the same aged women. This new trial aims to directly 
immunobridge to a population with demonstrated efficacy. Bridging to the nonavalent HPV 
vaccine would, in essence, bridge to a bridged population. This indirect approach would not be 
as definitive as bridging directly to the efficacy population. Yet, the plateau HPV16 antibody 
levels for Gardasil9 are not different compared to Gardasil and are 20% higher for HPV18 (56).  
Thus, if procurement of Gardasil is not possible, we will use Gardasil9 as the control vaccine.   
Selection of HPV 16 and HPV 18 antibody levels as outcome 
Although Gardasil additionally protects against the non-carcinogenic HPV types 6 and 11, which 
cause 95% of genital warts, this proposal only aims to evaluate the carcinogenic HPV types 16 
and 18. Approximately 50% of cervical cancer is caused by HPV16, an additional 20% by HPV18, 
and the remaining 30% by 10 other carcinogenic types collectively (7). While reducing the 
burden of genital warts is beneficial, our aim is to dramatically reduce cervical cancer incidence 
and mortality globally, hence the focus on the two most cancer-causing HPV types.  
Target populations 
Two groups will be investigated in the current proposal: 1) the efficacy population (women 
between the ages of 18 and 25, in which efficacy was previously demonstrated) and 2) the 
adolescent population (girls 9 to 14 years). The efficacy population was defined based on the 
trials documenting efficacy, which led to licensure. The FUTURE-II trial of GARDASIL established 
vaccine efficacy in 15-26 year-old females, the PATRICIA trial of Cervarix established efficacy in 
15-25 year-old females, and the Costa-Rica Vaccine Trial of Cervarix established efficacy in 18-
25-year-old women. Hence, we believe the oldest acceptable bridging population is 18-25 year 
olds.  
The World Health Organization (WHO) states that the primary target group in most of the 
countries recommending the HPV vaccination is young adolescent girls. Specifically, the WHO 
recommends a multi-age cohort during the first year of programmatic HPV vaccine 
implementation from 9-14 years with 2 doses of any of the licensed HPV vaccines. Hence, our 
objective is to establish vaccine efficacy for girls in this age range.  
Participant eligibility will not consider sexual history. Participants with evidence of prior HPV 
exposure, assessed retrospectively by HPV serology at baseline, will not be excluded from the 
study but will be excluded from the primary analysis.   

23 
 
Costa Rica as the study setting  
This study will be conducted using the established infrastructure used in the ongoing ESCUDDO 
study. 
This study will occur outside the catchment area for ESCUDDO. Therefore, we have identified 
twenty districts that combined, based on census data, have ~1600 girls and ~2300 women in 
the eligible age ranges for this study. These districts are in Guanacaste and we expect high rates 
of participation and follow-up. 
Specimen collection 
Blood is the only biospecimen that will be collected; it will be used for research purposes. Urine 
will be collected from participants 12 and older and tested for pregnancy at vaccination visits to 
assess eligibility for vaccination, but not stored.  
Eligibility criteria 
Within the selected PRIMAVERA catchment area (i.e., the 20 selected districts) all girls and 
women who are in the study age ranges will be invited to participate. Self-referral is permitted, 
as long as the potential participant is a resident of the catchment area. 
Inclusion criteria  
Potential participants will be deemed eligible if they are: 
• Female;  
• Aged between:  
o 9 and 14 years inclusive for Cervarix group; 
o 18 and 25 years inclusive for Gardasil group; 
• Living in the study area without plans to move outside the country in the next six 
months; 
• Able to communicate with study personnel; 
• Able and willing to provide a blood sample; 
• Willing to permit export of blood samples to the United States; 
• Willing to participate in the study and: 
o If Cervarix group and less than 12 years old, receive study information and be 
supported in study participation by at least one of parent (or guardian), who is 
willing to sign the informed consent document; 
o If Cervarix group and 12 years old or older, sign the informed assent and be 
supported in study participation by at least one parent (or guardian), who is 
willing to sign the informed consent document; 
o If Gardasil group, sign the informed consent.  
• In good health as determined by a medical history (physical exam will be conducted if 
necessary per the doctor’s criterion). 

24 
 
Exclusion criteria  
Potential participants will be excluded from enrollment if: 
 
• They have a diagnosis of an autoimmune, degenerative, or neurological disease without 
treatment or adequate control; a progressive or severe neurological disease; a genetic 
immunodeficiency; or any other serious chronic disease without treatment and / or 
adequate control that, according to the principal investigator or designee, for which 
vaccination is contraindicated (NOTE: Potential participants with these conditions can 
be included after consultation with the external medical advisor of the study or with an 
appropriate specialist); 
• They are allergic to one of the vaccine components, including yeast (if Gardasil group); 
• They have received immunoglobulins within 90 days preceding enrollment/vaccination 
visit; 
• They are unwilling to provide a blood sample; 
• Unwilling to permit export of blood samples to the United States; 
• They have a positive urine pregnancy test result; 
• They are pregnant;  
• They are planning to become pregnant. 
• The clinician determining eligibility in agreement with the principal investigator 
considers that there is a reason that precludes participation; 
• They have been vaccinated against HPV;  
• The participant or her parent/legal guardian, as applicable, does not have an 
identification document.  
Deferral criteria at enrollment: 
The enrollment visit will be deferred for either study arm if: 
• They have an acute disease that precludes vaccination (though vaccines can be 
administered to potential participants with a minor illness such as diarrhea and mild 
upper respiratory infection); 
• Are being evaluated by a clinician to rule out or confirm a diagnosis of a chronic disease; 
• The blood sample is not able to be collected. 
Contraindications to subsequent vaccination (Gardasil group) 
Participants in the Gardasil Group will not receive additional vaccine doses if they: 
• Present a significant hypersensitivity reaction following the first study vaccine 
administration; 
• Are diagnosed with any medically-documented immunodeficiency, autoimmune, or 
degenerative conditions; 
• Are diagnosed with any other newly acquired chronic condition which, in the opinion of 
the principal investigator, precludes further administration of the study vaccine; 

25 
 
• Present any SAE judged to be possibly, probably, or definitely related to study vaccine 
that may pose a risk to the participant should she receive another vaccine dose; or 
• Present any other significant reactions including severe pain, severe swelling, severe 
limitation of motion, persistent high fever, severe headache or other systemic or local 
reactions, which in the opinion of the study doctor and in consultation with the 
investigator preclude further administration of the study vaccine. 
 
Gardasil Group participants who do not receive the second or third vaccine dose will continue 
to be followed for the full length of study.  
Concomitant Medications/Vaccinations 
Concomitant medications administered during this study will be assessed in the context of AEs 
assessment only, as described in the adverse event reporting section.  
Outreach and Enrollment Visits for Both Cervarix and Gardasil Groups (0 months)  
We will use multiple methods to reach potential participants within the PRIMAVERA catchment 
area. The study will be publicized through attending community events, distributing outreach 
materials in commercial and residential areas, making announcements, and other means. 
Potentially eligible participants and parents/guardians of potentially eligible participants who 
are present during outreach activities will be informed about the study.  
If a potentially eligible participant (and her parent/guardian, as applicable), is interested, a 
participant study ID (PID) will be assigned and an enrollment appointment will be scheduled for 
her to attend a study clinic.  
During the enrollment visit at the clinic, the study will be explained in detail to the prospective 
participant and her parent/ guardian, as applicable. The informed consent and assent processes 
will be completed for those willing to participate. Consent for accessing the participant’s 
medical records from any medical institutions will be administered using separate consent and 
assent forms. If preferred, due to participant or parent’s/guardian’s needs, a visit may be 
requested to complete the informed consent/assent processes at home, prior to the 
enrollment appointment. 
After administration of the informed consent, the clinic team will proceed with eligibility 
determination. Among participants younger than 12 years old, the parent/guardian must be 
permitted to be present during all of the visit components. Among participants 12 years and 
older, the parent or guardian will be asked to leave the examination room while the pregnancy 
test is performed.  
Participants 12 and older will be asked to provide a urine sample for a pregnancy test, a 
component of the eligibility determination.  

26 
 
To continue the eligibility assessment, a clinician will assess the participant’s general health and 
rule out contraindications for receiving the vaccine, such as history of a chronic condition or 
allergies to any vaccine components. For girls 9-14 years old, if necessary, the clinician will 
consult with the participant’s parent/legal guardian. If the medical doctor considers necessary, 
based on the medical assessment, a physical examination will be performed to confirm good 
general health. If the medical assessment suggests an undiagnosed medical condition, the 
doctor may perform a directed physical examination to confirm a diagnosis, rule out a 
condition, or refer the potential participant for further medical evaluation.  
Finally, a blood sample will be collected. If the sample is not able to be drawn, the visit will be 
rescheduled. If the participant refuses the blood sample, she will be excluded from the study 
and will not receive the vaccine.  
An eligibility checklist will be completed during the eligibility determination phase of the 
enrollment visit to document whether the participant meets all the study and vaccine eligibility 
criteria.  
Once a participant meets the eligibility criteria and can be vaccinated, she will proceed with the 
vaccination procedures. After vaccination, participants will be observed for 15 minutes, per CDC 
guidelines, and as stated in the prescribing information for Cervarix and Gardasil. Appropriate 
medical treatment will be readily available in the rare event that an anaphylactic reaction 
develops. A real-time contact service (such as a toll-free number or text messaging system) will 
be staffed for the duration of the trial so that participants may contact the trial’s clinical team 
in case of emergency. 
Participants will be instructed to not become pregnant for about two months following 
vaccination and will be encouraged to use an effective birth control method if sexually active. 
This aligns with the labels for these vaccines, which state that the safety and effectiveness of 
the vaccines has not been established among pregnant women. Participants will be informed 
that while no risks have been observed for vaccination during pregnancy, the subject has not 
been well studied. For participants 12 years old and older, information about birth control 
methods will be provided upon participant request; however, we will not promote use of 
specific birth control methods.  
Gardasil Group Followup Vaccination Visits (2 and 6 months) 
Among women scheduled to receive Gardasil, the second and third vaccine visit will occur two 
and six months following the initial baseline visit. At these visits, vaccine eligibility will be 
assessed by the doctor or other qualified health professional, including assessment of any 
occurrence of serious adverse event reactions or newly chronic conditions diagnosed after the 
first vaccine.  

27 
 
As in the enrollment visit, a negative urine-based pregnancy test and report of no plans to get 
pregnant will also be required. Additionally, the health professional will complete a brief 
assessment to confirm that there are no vaccine contraindications and the participant is eligible 
to receive the second or third vaccine dose.  
If, during eligibility determination at the two- or six-month visit, a woman has a positive 
pregnancy test, she will be deferred, and her visit discontinued. If a woman is found to be 
ineligible for the second or third vaccine for another reason (i.e., related to vaccination only), 
she will be excluded from vaccination and will proceed with the other two- or six-month visit 
data collection procedures.  
If a woman meets the two- and six-month visit criteria to be vaccinated, she will proceed with 
vaccination. After vaccination, observation for 15 minutes will be completed and monitoring for 
adverse events performed as in the enrollment visit. 
Active Follow-up of Both Groups (Months 1, 12, 24, and 36) 
All participants will have follow-up visits every 12 months, and participants in the Cervarix 
group will have an additional visit one month after the vaccination. These visits will be 
conducted either in the home or a clinic setting. At each follow-up visit, the doctor or other 
qualified health professional will assess eligibility to complete the visit and collect updated HPV 
vaccination data. The 12-month visit for Gardasil group participants will include assessment of 
any occurrence of serious adverse event reactions or newly chronic conditions diagnosed after 
the final vaccination. Finally, blood will be collected from all willing participants. These visits can 
be completed at home. 
Home Visits During Follow-up 
All follow-up visits may be conducted at either the clinic or at home (at the convenience of the 
participant and/or her parent/guardian, as applicable), if an appropriate environment for 
performing blood collection is provided. The clinic team member who schedules the home visit 
will determine, based on an SOP created by the Investigators, whether the home setting is 
appropriate.  
Procedures and data collection  
Medical history  
At the enrollment visit, we will conduct a complete medical history (and physical examination if 
considered necessary by the clinician evaluating the eligibility) to determine the participants’ 
overall health status and eligibility for the study.  
Among younger participants, the medical history will initially be completed with the 
participant’s parent or guardian present to facilitate collection of information about any aspect 

28 
 
of her medical history with which the participant may not be familiar. After the first assessment 
of the medical history, the parent/guardian is asked to wait in the outer clinic area only if the 
participant is 12 years old or older. Then, the doctor will review the results of the pregnancy 
test.  
Along with determining whether a physical examination is necessary, the clinician will need to 
decide whether the parent or guardian should be present for the examination (if the participant 
is 12 years old or older). Participants under the age of 12 may remain with their parent/legal 
guardian during the entire visit. In any case, the nurse will always be present during physical 
examination. 
Should any condition be detected or suspected by the study clinician, we will inform the 
participant (and her parents if a minor and deemed necessary by the attending medical doctor) 
of the possible/diagnosed condition and refer the participant to her doctor. Specific 
SOPs/instructive and forms to conduct and document the enrollment medical history and 
general physical exam if performed will be used. 
Assessment of HPV vaccination outside the research study 
During the enrollment visit and the follow-up visits, participants will be queried about 
additional HPV vaccinations received outside the research study. To bolster and confirm self-
reporting, antibodies against HPV 6 and 11, the additional types included in the quadrivalent 
HPV vaccine that is provided as part of the Costa Rica National Vaccine Program, will be 
measured on a subset of participants as a biomarker to assess external HPV vaccination.   
Urine Samples (Vaccination Visits only) 
Along with the medical history, and before each vaccination, participants 12 and older will be 
asked to collect a urine specimen for a urine pregnancy test. Among participants 12 and older, 
only those with a negative pregnancy test will be enrolled.  
The participant will be directed to collect her urine in the bathroom with the doctor and nurse 
aide available to help her if she chooses. After the urine pregnancy test is completed, any 
residual urine sample will be discarded. 
Blood collection 
Blood specimens will be collected from all participants at every enrollment and follow-up visit. 
The purpose of these blood collections will be to monitor immune response to vaccination, to 
address study objectives. Less than 10ml of blood will be collected from all participants at each 
visit.  
In the clinic or home (in the case of in-home follow-up visit), during transport, and prior to 
processing, the samples will be stored according to standard operating procedures (SOPs) in a 

29 
 
cooler designed to safely and securely store and transport biological material according to local 
and international regulations. The blood will be stored at a temperature between 2˚C and 10˚C. 
Established SOPs provide instruction regarding the preparation and packing of the coolers, and 
proper operation and monitoring of the thermometers, and the actions needed to assure the 
required temperature for both storage and transportation.   
Vaccine Procedures  
Participants will be vaccinated intramuscularly in the deltoid muscle (upper arm), typically of 
the non-dominant arm, and will receive doses at enrollment for the Cervarix group, and, for the 
Gardasil Group, at enrollment and 2 and 6 months. 
All study vaccines will be stored at ~4°C per manufacturer instructions. Established SOPs will be 
followed to ensure proper chain of custody covering both custody of the pharmaceutical 
product and cold chain compliance.  
To assure adequate vaccine storage and custody, redundant power and refrigeration systems 
will be in place, in addition to temperature and access monitoring/recording systems. Vaccines 
exposed to damage or lack of custody will not be used in the trial and the event will be 
communicated to the Investigators. 
Blood preparation, storage and shipment 
We have experience collecting blood in Costa Rica from thousands of women over the last two 
decades as part of previous studies (57). Blood will be stored in cold boxes until transport to the 
local repository for preparation, freezing, and interim storage. Blood will be processed 
according to specified time requirements. For all blood samples, at enrollment and during 
follow-up, we will separate and store serum aliquots.  
Following processing, serum will be prepared and stored temporarily at the local repository, 
frozen at ultra-low or cryogenic temperature, until shipment to the NCI biorepository for long-
term storage. All serum aliquots will be shipped to the NCI repository. Further distribution will 
be done as needed, to collaborating laboratories for testing. A shipping and customs agent will 
be responsible for these shipments. MTAs between NCI and Costa Rican investigators 
complying with US and CR regulations will be in place before any shipment is conducted. 
Expected Adverse Events and Adverse Event Monitoring and Reporting 
There is considerable accumulated data on the safety of these FDA- and EMA-approved 
vaccines. Given the design of the trial, the study will lack reference rates of occurrence of 
specific events among unvaccinated women. Nevertheless, we will monitor all trial participants 
for adverse events (AEs) at the clinic for at least 15 minutes after vaccination with appropriate 
medical treatment readily available in case of a rare anaphylactic reaction. Adverse events (AEs) 

30 
 
occurring during this period will be documented and followed to resolution. In case participants 
present non-serious expected adverse events like site reactions (mild pain), dizziness, or 
headache they will receive recommendations and Paracetamol will be available.  
The consent and assent forms include a discussion of possible adverse events and will advise 
participants and their parents/guardians about what will happen if they experience an adverse 
event during the clinic visit. The consent and assent forms will also give guidance on how for 
participants to report adverse reactions that occur after the participant leaves the visit. The 
consent forms explain the study’s insurance coverage and that, per Costa Rican Human 
Research Law, the study may pay for the care of conditions determined to be related to the 
study vaccines or procedures.  
In addition, following each vaccination, participants will be told that they should report to study 
staff any serious problems they have in the month after each vaccine administration that 
require medical attention, whether they think the problem is related to vaccination or not.  
Finally, participants will be actively queried at the study visit that follows each vaccine 
administration regarding SAEs that occurred after vaccination. Additionally, Cervarix group 
participants will be contacted at 6 months, either by phone or personally to inquire about 
vaccine reactions and to maintain contact to promote compliance with follow-up visits. In 
either case, the clinician will probe for adverse events by asking the participant whether she has 
been diagnosed with an illness, disabled for more than one day, or hospitalized since her last 
visit. These questions are included in the eligibility screener at the second and third vaccination 
visit, and the 12-month visit for the Gardasil Group, and in the six-month contact script for 
Cervarix group.  
For the full duration of the trial, AEs spontaneously reported by study participants will be 
documented. All deaths will be documented irrespective of timing between the occurrence of 
the event and vaccination.  
All the adverse events, serious and not serious, reported by participants as part of active 
monitoring during the visit after vaccination completion (i.e., the 6-month phone contact for 
Cervarix group participants, and the 2-, 6- and -12-month visits for Gardasil group participants), 
or as part of passive monitoring throughout the duration of the study (i.e., reported 
spontaneously by the participant) will be documented. All events that are not resolved at the 
time of reporting will be followed up to document their resolution; if the SAE results in a 
chronic condition, it will be followed until medical management of the condition is 
documented. Should the event result in hospitalization, or otherwise require hospital record 
review according to the criteria of the Principal Investigator or designee, trained staff will 
review the medical records at the hospital, if necessary, and will extract relevant information to 

31 
 
clarify the evolution, diagnosis, treatment and resolution of the event. In a separate informed 
consent/assent form, participants and their parents/guardians, as applicable, will be asked to 
provide authorization for review of hospital charts if necessary. 
Non-serious AEs directly related to study procedures other than the vaccine (e.g., blood 
collection) also will be documented and followed to resolution. 
The Costa Rica Principal Investigator will report SAEs to the responsible regulatory authorities in 
Costa Rica, and to the NCI Principal Investigator. The reporting of AEs to regulatory entities in 
the United States will be the responsibility of the NCI Principal Investigator. Procedures for 
reporting AEs in the United States will be determined according to the NCI’s Ethical Review 
Panel (ERP) guidance.  
The Costa Rica Principal Investigator will report AEs to the Costa Rica IRB as stipulated in the 
regulation of the Human Subject Research Regulatory Act (Law 9234). Additionally, the Costa 
Rica Principal Investigator will report all AEs related to vaccination to the Pharmacovigilance 
office of the Costa Rica Minister of Health.  
The DSMB will also monitor AEs for safety. The DSMB will review AEs according to their 
reporting and meeting schedule, as specified in the DSMB Charter. 
Protocol Deviation Monitoring and Reporting 
Serious and non-serious protocol deviations will also be reported to the Costa Rica and NCI 
IRBs. The Costa Rica Principal investigator will report protocol deviations to the local IRB in 
interim reports in compliance with IRB and national regulations.  
It will be the NCI Principal Investigator’s responsibility to report to entities in the U.S. 
Procedures for reporting of serious and non-serious PDs will be determined according to the 
ERP guidance.  
Internal and external trial monitoring will be conducted. 
Data collection and electronic records 
Electronic case report forms (eCRFs) will be used in lieu of paper case report forms. Data will be 
collected using devices with custom-developed applications and programs. Real time edits and 
validation checks will be implemented into the eCRF system. All data that are considered 
personally identifiable will be stored encrypted, if stored locally on a cellular device. All data 
that are sent from any electronic device to the main servers will be sent encrypted. Physical 
security measures will be placed around all devices that are to be used to collect data 
electronically at the clinic. The main servers will be stored in a restricted access location and 
will only be able to be accessed by systems administrators and other authorized IT personnel.  

32 
 
A strict and rigorous permission-based editing system will be implemented to allow only certain 
personnel with specific permissions to modify data once that data has been committed to the 
main database. A full audit trail will be kept on all data modifications, which will include the 
user making the change, date and time, old value, new value and reason for the change. The 
modifications will be viewable to monitoring personnel in order to ensure full traceability. 
Serology  
HPV binding is measured by ELISA. The quantity of the HPV16 and 18 IgG antibody response 
measured by VLP ELISA will be utilized to address immunogenicity endpoints.  
To further define the primary analytic cohort, HPV6 and HPV11 antibodies will be measured by 
VLP ELISA in a subset of participants to confirm, beyond self-report, that the according to 
protocol analytic cohort includes only girls who did not receive external HPV vaccination 
through the national program, which uses quadrivalent vaccine (HPV 6, 11, 16, 18).  This will 
assure that girls in the single-dose HPV vaccination arm did not get additional doses.  
HPV neutralization is measured by SEAP-NA. Antibody levels measured by ELISA will be 
compared to neutralizing antibody levels measured by the pseudovirion-based neutralization 
assay, SEAP-NA. This nested study will select 36-month serum samples from a random set of 
100 18-25 year old women who are HPV 16/18 seronegative at baseline and a random set of 
100 9-14 year old girls who are HPV 16/18 seronegative at baseline, and then evaluate the HPV-
16 and HPV-18 antibody levels in each of those 200 samples using both ELISA and SEAP-NA. For 
each HPV type, we will perform a Deming regression comparing log(ELISA) levels with log(SEAP) 
levels, where log(ELISA) is the dependent variable and log(SEAP) is the independent variable. 
We will consider ELISA as an acceptable assay for the primary endpoint if the 95% confidence 
intervals of the regression slope excludes 1-Δ and 1+Δ for both HPV types, with Δ = 0.3. If the 
true slope is b=0.9, the variance of the measurement error for both log(ELISA) and log(SEAP) is 
σ2 = 0.4, and the total variance of log(SEAP) = 1.9, then the power to achieve this serology 
objective is 0.94. Preliminary data suggests that 0.9 < b < 1 and 0.10 < σ2 < 0.45. The power for 
other combinations of sample sizes, slopes, and variances are provided in Table 3. Note the 
power is significantly reduced if we consider each group of females separately. Furthermore, 
we will estimate the intra-assay measurement error by measuring 30 samples, 15 from 9-14 
year old girls and 15 from 18-25 year old women, twice on each of the assays. Therefore, for 
the purposes of the serology study, the total number of samples run on each assay will be 
230. If we do not achieve the serology objective, we will evaluate the data with partners at 
regulatory agencies to determine whether ELISA can be still be considered as an acceptable 
assay for measuring the primary endpoint.  

33 
 
Table 3: Power calculations. The table lists the power to demonstrate that the 95% 
confidence interval for the slope describing the relationship between ELISA and SEAP-NA 
measures, on the log scales, excludes 0.7 and 1.3 for the specified sample sizes (N = 50, 100, 
150, or 200), slopes (b = 0.9, 1.0), and variances (σ2 = 0.25, 0.45).  
 
b = 0.9 
 
N = 50 
N = 100 
N = 150 
N = 200 
σ2 = 0.25 
0.54 
0.92 
>0.99 
>0.99 
σ2 = 0.45 
0.12 
0.48 
0.73 
0.88 
 
 
b = 1 
 
N = 50 
N = 100 
N = 150 
N = 200 
σ2 = 0.25 
0.85 
>0.99 
>0.99 
>0.99 
σ2 = 0.45 
0.17 
0.79 
0.97 
>0.99 
 
Efforts to maximize participation and retention  
Efforts aimed at promoting participation during enrollment, and minimizing withdrawal and 
losses during follow-up, include:  
• Intensive training on communication skills for staff who will be conducting outreach 
activities to potential participants and their parents/guardians, as applicable. 
• Multiple efforts at different times will be made to contact potential participants and their 
parents/legal guardians.  
• Flexible clinic hours to accommodate scheduling needs of participants and 
parents/guardians. 
• The option to complete the informed consent/assent procedures and/or follow-up visits 
(i.e., the 24-month and 36-month visits) in the home in order to accommodate 
participant’s and parent’s/guardian’s scheduling needs (provided that a suitable space is 
available, according to established SOPs).  
• Free phone line for potential participants to call and request or change an appointment. 
• Reminder phone calls and text messages about appointments. 
• Reimbursement for transportation costs, if needed, and, if requested, transportation to 
the clinic in a study vehicle. 
• Exclusion of participants who report (or whose parents report) they have definite plans 
to move out of the country within six months of enrollment.  
 
Efforts described above should minimize withdrawals from the study prior to the completion of 
36 months of follow-up. In the event that a girl or woman withdraws from the study, the reason 
for withdrawal will be documented.  

34 
 
Discontinuation of vaccination (i.e., withdrawal from vaccination) will not constitute grounds 
for withdrawal from study follow-up. The occurrence of an AE will also not necessarily 
constitute grounds for withdrawal.  
Approval of the exiting of participants from the study 
The study is proposed to last three years after initial vaccination for each participant. Once the 
last study visit is completed, the participant chart will be reviewed to ensure that all serious 
adverse events are resolved or, if chronic conditions are present, that they are under medical 
care. The participant will then be closed out from the study. As mentioned above, if the GMT 
for 1 dose at the group level is inferior to that of 3 doses based on the 36-month analysis, all 1-
dose girls will be invited to receive the 2nd dose of an HPV vaccine. Previous trials documented 
that delayed administration of a booster dose did not diminish the subsequent increase in 
antibody levels (58). 
ANALYSIS PLAN  
Primary Analytical Cohort 
To evaluate the non-inferiority objective in the trial, an According to Protocol (ATP) analytical 
cohort will be defined. The ATP cohort for a specified HPV type (i.e. HPV 16 or HPV 18) and 
timing (i.e. 24-month or 36-month) are the participants who satisfy the following set of 
requirements: 
• Received the correct number of doses within the predefined ATP vaccination windows. 
Denoting T1 as the time of the first vaccination, the analytical window for the second 
vaccination is between T1+30 days and T1+90 days. Denoting T2 as the time of the 
second vaccination, the analytical window for the third vaccination is between T2+90 
days and T1+360 days.  
• Not seropositive at baseline for the specified HPV type.  
• Blood collected within the predefined ATP follow-up windows.  
• No HPV vaccination outside of the study prior to blood collection. The participant did not 
report receiving an HPV vaccine and, among those tested, did not have HPV6 and HPV11 
vaccine-level antibodies.  
The cutoff for HPV16 seropositivity is greater than 1.41 international units (IU)/mL and for 
HPV18 is greater than 1.05 IU/mL. All serological measurements will be presented in 
international units Restricting to HPV16 or 18 seronegatives using this cutoff also aligns the 
Gardasil cohort in this study with the efficacy cohort used in the licensure of Gardasil (Future I 
and II) (12).  

35 
 
Immunogenicity Endpoint  
The four endpoints for the primary objective are HPV-16 and HPV-18 specific antibody results 
obtained from blood specimens (serum) collected at the month 24 (interim) and 36 (primary) 
month visit (i.e. 4 endpoints = 2 HPV types x 2 time-points). All antibody levels are measured by 
HPV type-specific ELISA. Two additional endpoints, HPV-16 and HPV-18 antibody levels measured 
from serum collected at the 1-month visit and the 1-year visit, will also be included to achieve 
Secondary Objective 4. 
Analysis  
We will estimate the GMT ratios for four primary endpoints: HPV-16 and HPV-18 at 24 and 36 
months.  
• We define 𝑅𝐻(𝑡) = 𝐺𝑀𝑇1
𝐻(𝑡)/𝐺𝑀𝑇3
𝐻(𝑡) to be the GMT ratio for HPV-H (i.e. HPV-16 
or HPV 18) at time point t (i.e. 24 or 36 months) comparing type-specific antibody 
levels in 9-14 year old girls receiving 1 dose of Cervarix to levels in 18-25 year old 
women receiving 3 doses of Gardasil. 
• We will estimate 𝑅𝐻(𝑡) by fitting a generalized estimating equations (GEE) model 
where the dependent variable is the log-antibody level and the independent variable 
is study-group. Denoting the estimated coefficient for study-group by 𝛽̂𝐻(𝑡), we 
define our estimate of the desired ratio by 𝑅̂𝐻(𝑡) = exp (𝛽̂𝐻(𝑡)). In the model, we 
will group girls into clusters of full siblings and use an exchangeable correlation 
matrix. 
• We will estimate the two-sided 99% confidence interval of the GMT ratios for HPV-
16 and HPV-18 at 24 months and the two-sided 96% confidence intervals of the GMT 
ratios for HPV-16 and HPV-18 at 36 months. 
• Denoting the corresponding robust standard error of 𝛽̂𝐻(𝑡)  by 𝜎̂𝐻(𝑡), we define the 
two-sided (1-α)% confidence interval (CI), (𝑅̂𝛼2
⁄
𝐻(𝑡), 𝑅̂1−𝛼2
⁄
𝐻
(𝑡)), by exp (𝛽̂𝐻(𝑡) ±
𝑧1−𝛼2
⁄ 𝜎̂𝐻(𝑡)), where  𝑧1−𝛼/2 is the 1-α/2 quantile of a normal distribution. 
We will declare non-inferiority if min (𝑅̂0.005
16
(24), 𝑅̂0.005
18
(24)) > 0.67 or, upon failure of that 
criterion, min (𝑅̂0.02
16 (36), 𝑅̂0.02
18 (36)) > 0.67. Note, under the null hypothesis that both 
min(𝑅16(24), 𝑅18(24)) < 0.67 and min(𝑅16(36), 𝑅18(36)) < 0.67, the probability of 
declaring non-inferiority or the Type I error rate is less than 0.025 for HPV-16 and HPV-18 
endpoints, respectively. 

36 
 
Because the national vaccination program has been initiated, there is the potential that some 
girls enrolled at ages 9-10 years may have received an additional vaccination dose but have 
failed to report it. Therefore, we plan to conduct the following secondary/sensitivity analysis. 
We will compare GMTs, distributions, and seroconversion rates for HPV-16 and HPV-18 
antibodies, assessed at 24 and 36 months after initial vaccination, between 11-14 year old girls 
receiving 1 dose of Cervarix and 18-25 year old women receiving 3 doses of Gardasil. We will 
also calculate the GMT ratios, comparing girls 9-10 years old at vaccination with girls 11-14 
years old at vaccination, and their 95% confidence intervals for HPV-16 and HPV-18 antibody 
levels at 1 month and 1-year post-vaccination. Additionally, we will test for HPV6/HPV11 
antibodies in a subset of participants who are at-risk for external HPV vaccination or show 
serologic evidence of additional HPV vaccine doses. 
Power and Sample Size Considerations 
We calculate the power to show non-inferiority at 24 months and the power to show non-
inferiority at 36 months given that we enroll 520 women 18-25 year old and 347 girls 9-14 year 
old. Specifically, we assume that 𝜎̂ = 1.38, 20% of all 18-25 year old women do not receive all 3 
doses, 100% of 9-10 year old girls (i.e. worst case scenario) receive a vaccination outside the 
study, 20% of all participants are lost-to-follow up, 10% of the 18-25 year old women are 
seropositive at baseline, 5% of the 11-14 year old girls are seropositive at baseline, the common 
true ratio, R, for HPV-16 and HPV-18 is 1, and the non-inferiority threshold (t) is 0.67, in 
accordance with the recommendation of the WHO Expert Committee on Biological 
Standardization(60). Based on these assumptions, we will have 65% power to demonstrate 
non-inferiority at 24 months and 84% power to demonstrate non-inferiority at 36 months. 
Power calculations for other values of R and t are listed in table 4. Note the power reported for 
36 months is Pr (min (𝑅̂0.02
16 (36), 𝑅̂0.02
18 (36)) > 𝑡), a lower bound for the probability of 
rejecting non-inferiority at either the 24- or 36- month analysis. In the more optimistic scenario, 
where 9-10 year old participants do not receive vaccinations outside the study, our power 
increases to 78% at 24 months and 91% at 36 months.   
For the potential secondary analysis, we would calculate the GMT ratios, comparing girls 9-10 
years old at vaccination with girls 11-14 years old at vaccination, and their 95% confidence 
intervals for HPV-16 and HPV-18 antibody levels at 1 month and 1 year post-vaccination. 
Assuming that enrollment includes 173 girls 9-10 years old and 347 girls 11-14 years old, 5% of 
all girls are seropositive at baseline, 10% of all girls do not return for the 1 month visit, R = 1, 
and 𝜎̂ = 1.38, the expected length of the 95% confidence interval for 𝑅̂𝐻(1) is 0.55 (i.e. if 
𝑅̂𝐻(1) = 1, the expected 95% confidence interval would be 0.76 – 1.31). 

37 
 
Table 4: Power calculations. The table lists the power to demonstrate non-inferiority at the 
24- and 36-month analyses for a non-inferiority threshold of 0.67 and three values of the 
common true ratio (R=0.8, 1.0, 1.2), with other parameters set at the values described in the 
text.  
 
Threshold = 0.67 
 
Power 
(24 months) 
Power 
(36 months) 
R = 0.8 
0.02 
0.09 
R = 1.0 
0.65 
0.84 
R = 1.2 
>0.99 
>0.99 
 
HUMAN SUBJECTS PROTECTIONS 
This trial will be conducted in compliance with regulations for human subject research in effect 
in Costa Rica (Costa Rican Human Research Law 9234) and the United States (The Belmont 
Report & Title 45 Code of Federal Regulations, Part 46, Protection of Human Subjects, and 
Declaration of Helsinki). The principles of respect for persons, beneficence, and justice will be 
paramount in planning our trial with all the necessary considerations related to inclusion of 
minors.  
In additional to specialized training associated with their roles, all study staff involved in study 
interactions with participants, their confidential data or samples (e.g., outreach workers, clinic 
staff, relevant systems staff, laboratory staff, sub-investigators and principal investigators), will 
complete GCP training in accordance with the requirements of the Costa Rican Ministry of 
Health, which authorizes them to conduct human research. They will sign a confidentiality 
agreement as part of their contract.  
Institutional Approvals  
The initial protocol was approved by the Senior Advisory Group (SAG) Committee of the 
Division of Cancer Epidemiology and Genetics on September 17, 2018. All comments and 
suggestions which came out of the SAG review were incorporated. In addition to the NCI IRB, 
we will obtain IRB approval from a Costa Rican Ministry of Health-approved institutional review 
board designated by Fundación INCIENSA under an appropriate FWA.  
Justification for exclusions and dose schedule 
Most vaccine programs seek to immunize 9- to 12-year-old girls. However, since individual level 
benefit has been documented at older ages, catch up vaccination through the age of 26 years is 
also recommended in many countries. Our study will enroll women up to age 25. 

38 
 
Although HPV vaccination has been shown to be equally effective in boys, this study will focus 
only on females because, in LMIC, women bear the overwhelming burden of HPV-driven 
disease. To our knowledge, none of these countries is considering gender-neutral vaccination 
strategies. Thus, it is important to maximize the amount of information on the target 
population.  
Only girls and women with a negative pregnancy test will be enrolled as study participants. If a 
potential participant has an equivocal or positive pregnancy test, she will be excluded from 
enrollment and vaccination according to the vaccine use and recommendations approved by 
the Ministry of Health. This is according to the labels for these vaccines, which state that the 
safety and effectiveness of the vaccines has not been established among pregnant women.  
Safeguards for vulnerable populations  
For women who are 18 or older, only a signed consent from the participant is required. For 
participants 12-14 years old, permission from one of the parents or legal guardian will be 
required, along with a signed assent from the girl. For girls younger than 12 years old, 
permission from one of the parents or legal guardian will be required, along with the girl’s 
verbal agreement to participate. This is in accordance with the Costa Rica law and the NCI IRB 
policy for consenting minors. A girl’s failure to give assent is binding, and girls who are 
incapable of providing assent will be excluded. The permission will be documented by obtaining 
a signed consent form from a parent or legal guardian according to regulation, along with a 
signed assent form from the girl.  
Different forms will be used for younger participants, one for ages 9-11 and one for ages 12-14. 
The length and complexity of the language in these forms has been reduced and simplified to 
aid comprehension but includes all the information required by law. In addition to the different 
language and content used in these forms, the way that willingness to participate is provided 
will differ. This is according to Article 17 of Costa Rican Regulatory Law of Biomedicine 9234, 
which specifies that signed informed assent is required starting at age 12. Girls 12-14 will be 
asked to provide written assent. Girls 9-11 will be administered a study information document 
and their verbal agreement to participate will be recorded in the study management system.  
The result of the pregnancy test will be provided to the participant only, in private. Participants 
age 12-14 years old who have a positive pregnancy test result will be notified as follows: 
• The clinician will discuss this with the participant, and in discussion with the participant, 
the clinician will select one of the following options: 
o Have the doctor inform her parents, 
o Inform her parents by herself, or 
o Not inform her parents at all. 

39 
 
• In all cases, the doctor will make sure the participant understands that PANI will be 
informed, that PANI will follow-up with her, and that ACIB will follow-up with her and/or 
PANI to ensure she receives PANI follow-up. 
 
These procedures will be followed in order to avoid exposing participants to risk due to 
disclosure of pregnancy test results to their parents in cases where this represents a risk of 
aggression and in order to protect the participant’s rights to knowing her information and to 
preserve confidentiality. This policy is consistent with Costa Rican regulations (Código de la 
Niñez y la Adolescencia - Law 7184, Código Procesal Penal - Law 7594, and Ley General de 
Protección a la Madre Adolescente - Law 7735).  
The topic of safety during pregnancy has been evaluated using CVT data (61, 62). Results from 
these analyses have been reassuring with respect to the safety of vaccination during pregnancy. 
Although we will conduct urine pregnancy tests in participants 12 and older to rule out 
pregnancy prior to vaccination, in the event a participant is vaccinated while pregnant (i.e. due 
to a false negative pregnancy test) or becomes pregnant after the first dose, the second and 
third doses will be deferred until after pregnancy resolution (depending on whether the 
applicable study visit is still open). No special follow up is required and participants who 
become pregnant will continue in the study and will be followed for the full 3 years.  
We note, however, that the expected number of vaccine exposures in pregnant women will be 
small and no internal control group exists in our study, so the information obtained from this 
study is expected to provide limited new information above and beyond what has already been 
published regarding adverse events associated with immunization during pregnancy.  
All participants with positive pregnancy test results at the enrollment or at one of the other 
vaccine visits, as applicable, regardless of their age, will be informed about the need for pre-
natal care and will be referred to the appropriate health care service for prenatal care and to 
the PANI for social follow-up in the case of the Cervarix group, which includes ensuring access 
to pre-natal care due to the high-risk nature of pregnancy during adolescence. We will support 
the participant (i.e., providing information, education, appointments coordination, etc.) in 
obtaining prenatal care at the appropriate health care service. Follow-up contact will be 
conducted to determine if the participant is attending prenatal care and to emphasize its 
importance. Additionally, if the doctor believes the participant needs immediate support, the 
doctor will contact by phone a study psychologist, who will guide the clinician on how to 
proceed, including immediate referral to appropriate institutions that provide support. 
The Costa Rican government implemented school-based HPV vaccination with two doses of the 
quadrivalent vaccine in fourth grade girls (typically age 10) in 2019. Consequently, a subset of 

40 
 
study participants may receive, in addition to the one dose of Cervarix as part of the trial, one 
or two additional doses of Gardasil outside the study. Studies on mixed-dosing schedules of the 
HPV vaccines have confirmed its safety and immunogenicity (58, 63-65). In particular, 
researchers in Quebec conducted a trial evaluating the immunogenicity and safety of a mixed 
vaccination schedule randomizing girls into the following three arms: 1) one dose of the 
bivalent HPV vaccine followed by one dose of the nonavalent HPV vaccine, 2) one dose of the 
nonavalent HPV vaccine followed by one dose of bivalent HPV vaccine, and 3) two doses of the 
nonavalent HPV vaccine (64). From this work, mixed HPV vaccination schedules were 
documented to be sufficiently immunogenic (all participants were seropositive) and have an 
acceptable safety profile. The safety of a mixed dosing schedule is further supported by the 
recommendations of the CDC Advisory Committee on Immunization Practices, which states: “If 
vaccination providers do not know or do not have available the HPV vaccine product previously 
administered, or are in settings transitioning to 9vHPV, any available HPV vaccine product may 
be used to continue or complete the series for females for protection against HPV 16 and 18” 
(66). 
The study team is sensitive to the issues associated with the vulnerable younger population of 
this study, in terms of parental consent and participant assent, and procedures that may cause 
discomfort. Together with the DSMB, we will conduct periodic reassessments of the protocol 
with regard to these considerations. 
Consent process and documentation  
The study will conduct community outreach in commercial and residential areas, including 
door-to-door contacts, to identify households with potential participants, and approach them. 
If the participant and parent/guardian, as applicable, are present and available to discuss the 
study, the objectives of the study will be explained to them. When possible, potentially eligible 
participants will be scheduled for a clinic appointment at a time that is convenient for the 
participant and her parent/guardian, as applicable, and outreach materials will be left with 
them to review and discuss.  
In order to accommodate the participant or parent’s/guardian’s scheduling needs, members of 
the study staff who are trained to administer the informed consent forms will be available to 
complete the informed consent and assent administration procedures at the home. In such 
cases, even if a younger potential participant is unable to ask questions privately in the home, 
she will have the opportunity to ask questions, and withdraw or modify her assent status when 
she comes to the clinic to complete the enrollment visit. 
Formal administration of the consent forms will typically take place at the clinic appointment. A 
trained consent/assent form administrator will welcome the participant and parent/guardian (if 

41 
 
applicable), seat them, and invite them to view a video on a tablet that provides a visual 
presentation of the assent and consent form contents; there will be a version for girls, a version 
for parents/guardians, and a version for adult participants. The team member administering 
consent will then present the consent, assent, and information forms, as applicable, and assist 
the potential participant and parent/guardian, as applicable, to read and review the content. 
The consent administrator will address any questions the participant and her parent or 
guardian may have and then assist them to complete the informed consent/assent and 
signature procedures. A trained consent/assent form administrator will be present at all times 
at the study clinic to address questions and concerns and keep study subjects informed about 
the study procedures and progress. The study clinician will be available to answer more medical 
questions. As described, we will use different forms according to the age of Cervarix group 
participants, as well as a consent form for parental consent of Cervarix group participants, who 
are all younger than 18; girls who become 12 during the follow-up must sign an informed 
assent. The Gardasil group will all be adults and thus require only a consent form for adult 
participants. 
In accordance with Costa Rican research regulations, a witness signature will be collected. This 
witness will not be a member of the study staff and will not be given a copy of the 
assent/consent form, although a copy will be given to the girl and parent/guardian. The format 
of the witness signature implemented on the consent/assent forms will be as required by these 
regulations (Article 10 of Law 9234). In practice, the witness is usually a family member/parent 
of another participant, or a neighbor, not related to the research team, and who will have not 
access to the confidential information of the participant (Regulation of Law 9234: - Article 8, 
part a. “In all biomedical research in which human beings participate, except for those indicated 
in articles 7 and 12 of Law No. 9234, the researcher must obtain informed consent, individual, 
voluntary, express, specific, written and signed or with the fingerprint of the participant or his 
legal representative on all sheets, and an impartial witness on the final sheet. In the case of the 
legal representative: A copy of documentation should be included where the right to legal 
representation is registered.”; Article 8, part f. “The process of signing informed consent must 
be made in the presence of an impartial witness, who may be a family member, selected by the 
participant or his/her legal representative, without contact with the research team, 
researchers, Sponsors, OACs and ICOs. The witness must be a person with sufficient capacity to 
understand the scope of his performance, will not have access to the confidential information 
of the participant, nor be present when it is analyzed.”) 
The consent forms will include separate yes/no fields for indicating consent or assent for long 
term storage of samples and data sharing, as well as contact for future studies. Consent for 
accessing the participant’s medical records from any medical institutions will be administered 
using separate consent and assent forms.  

42 
 
Subject privacy and confidentiality of data, including plans for sharing, future use, and 
disposition of study materials/biologic specimens  
Study procedures will be conducted in an enclosed private environment. All study staff will be 
trained in ethical conduct of research. We propose to retain the data and specimens from the 
study for a period of up to 50 years. However, study subjects have the right to withdraw at any 
time by informing a member of the outreach or clinic staff, calling the central study number, or 
contacting the local PI to request consent withdrawal. Upon receiving such a request, study 
staff will document the consent withdrawal, including the source of the request (e.g., 
participant, parent/guardian, or PI/Designee); the specific components of consent withdrawal 
(e.g., Biobanking, Medical Chart Review, and Contact for Future Studies); and the reason for 
withdrawal, if known. If the participant wants to withdraw from the study this will be 
documented as a study discontinuation. All consent withdrawals and study discontinuations are 
reviewed by the PI or a designee and, if considered necessary, an additional contact will be 
attempted to provide missing information or clarify issues before approving the withdrawal. As 
applicable, the requestor’s data will be excluded from the study and/or biological samples be 
discarded for future analyses.  
Only informed assent and consent forms will be kept in hard copy in locked cabinets, with 
access limited to study personnel. Electronic data will be stored in a password protected 
centralized database, and access to these data will be granted to only authorized study staff 
and authorized IT personnel. Whenever these data are transferred out of the centralized 
database for analysis purposes or any other purpose, all personally identifying information that 
is not necessary for analysis will be removed, and transfer of the data will be done using a web-
based secure file transfer service.  
Information provided by study participants will never be used to directly identify them by NCI 
or outside investigators. Additionally, each study participant will be assigned a unique study 
number and a link will be maintained with personal identifiers. Information on personal 
identifiers will include name, date of birth, and a unique national ID number. This information is 
stored only for the purposes of subject follow-up and will not be shared with NCI. 
Serum collected as part of the study will be retained for use in the future. Such use will be 
restricted to assays required to address questions related to immune response to HPV and HPV 
vaccination. Note that future of use of serum does not include any genetic analyses. The 
participant or her parent or legal guardian can refuse bio-banking of biospecimens without 
affecting her participation. 
Plan for return of individual study results to participants  
Results from the molecular assays will be used only for research purposes.  

43 
 
Evaluation of anticipated benefits and risks/discomforts  
Participants will receive the benefit of HPV vaccination. Additionally, women who are within the 
age range to receive screening for cervical cancer in the Costa Rica healthcare system, will 
receive education concerning the importance of screening for secondary prevention. 
The risks associated with this research are believed to be minimal. Both vaccines are approved 
products and known risks include injection site reactions (mainly pain), dizziness, and headache. 
A series of reviews of safety data have been conducted by both the Institute of Medicine (IOM) 
and the WHO Global Advisory Committee on Vaccine Safety (GACVS). These reviews continue 
to affirm that no safety issues that would alter the recommendation for use of the HPV vaccines 
have been found (25, 67, 68). 
We will monitor all study participants for adverse events (AEs) at the clinic for at least 15 
minutes after vaccination with appropriate medical treatment readily available in case of a rare 
anaphylactic reaction.  
While no risks have been observed for vaccination during pregnancy (61, 62), if a potential trial 
participant is planning to become pregnant, or has an equivocal or positive pregnancy test, we 
will defer her enrollment and vaccination according to the vaccines use and recommendations 
approved by the Costa Rican Ministry of Health. This is according to the labels for these 
vaccines, which state that the safety and effectiveness of the vaccines has not been established 
among pregnant women. Pregnancy test results will also be handle as described above to 
protect participants (See section “Safeguards for vulnerable populations”). 
HPV vaccination has no effect of behavior or sexual risk taking. There are extensive data from 
more than 20 studies that occurred in multiple world regions, and across a broad age range 
including girls and women (69). Outcomes in these studies included measured changes in sexual 
behaviors such as age at sexual debut, risky sexual behaviors, use of condoms and 
contraception. More importantly, clinical indicators such as rates of sexually transmitted 
infections, HIV, and pregnancy have also demonstrated no differences between vaccinated and 
unvaccinated populations. In the event that one dose does not elicit strong immune response 
and girls do not obtain adequate protection, data suggest that these vaccinated girls/women 
would not be at higher risk of HPV acquisition due to increased sexual risk taking.  
From venipuncture, risks include a small bruise and the minor pain associated with a needle-
stick. Every effort will be made by the qualified phlebotomists to be hired by the Costa Rican 
collaborators, to minimize the pain from needle-stick and subsequent bruising or bleeding.  

44 
 
Compensation 
Study participants will not be compensated for their participation and time, but they will be 
reimbursed for transportation costs. Participants will be reimbursed their actual costs, which 
will depend upon where they travel from and whether they came accompanied or 
unaccompanied by additional persons such as parents or other family members. The actual cost 
is determined by self-report and staff knowledge of the standard local fares (e.g., local bus 
fares), and expected taxi fares, for example from bus station to home. Finally, if the participant 
is present at the clinic during breakfast, lunch, or dinner time, food will be offered; throughout 
the day, coffee, water, and light snacks also will be available. 
ETHICAL CONSIDERATIONS AND TRIAL OVERSIGHT 
The trial will make use of several methods for data and safety monitoring, and oversight. 
Data and Safety Monitoring 
A Data Safety Monitoring Board (DSMB) will meet during the vaccination phase of the trial and 
as required during the follow-up phase. After each review, members of the DSMB will 
summarize their findings and provide recommendations to the NCI Principal Investigator, the 
DCEG Scientific Director, the Costa Rican Principal Investigator, and the NCI Ethics Review Panel 
(ERP) and Costa Rican IRB. Should the NCI or local IRB have any concerns regarding trial 
progress or participant safety, these will be addressed directly to the DSMB with a copy to the 
Costa Rica and NCI Principal Investigators. Full communication between the ERP, IRB, and the 
DSMB is essential and will be the responsibility of the NCI Principal Investigator. A DSMB 
charter document will detail the charge to the committee, membership, implementation and 
reporting plans. 
Delivering trial results to participants and actions if study shows one dose failure. 
As previously mentioned, the efficacy of a single dose will only be known towards the end of 
the three-year follow-up. At the conclusion of this study, if single-dose vaccination is 
immunologically inferior compared to the standard arm in this study, the investigators will 
inform single-dose recipients, and offer the second dose of the HPV vaccine.  
In the event that delivery of vaccine information to participants is necessary, we will seek the 
permission of the local IRB to make contact with participants who have withdrawn from the 
study. This authorization is required according to GCP and laws governing research in Costa 
Rica, which otherwise bar study personnel from making contact with this population. Based on 
our past experiences with the CVT cohort, we estimate that fewer than 2 percent of 
participants will be truly lost to follow-up (i.e., we cannot find or contact them), so we are 
confident we will be able to follow-up with most participants, as needed, provided that we have 
the IRBs authorization. Nevertheless, as an added precaution, we will include language in the 

45 
 
consent and assent form urging all participants to retain the consent form so that they are able 
to contact the study in the future even if they discontinue participation. At the point of 
withdrawal, we will also emphasize these instructions and will provide participants who are 
withdrawing from the study with whatever information we can at the time they withdraw, if 
possible (i.e., if they agree to receive the information).  
Working Group 
In addition to the DSMB, an independent Scientific Working Group (WG) will be established by 
NCI to advise the study investigators and the Division Director on scientific and policy issues 
surrounding the trial. The WG membership will consist of individuals with expertise in areas 
relevant to the conduct of the trial, including clinical trials, epidemiology, biostatistics, virology, 
vaccinology, immunology, medical ethics, and health advocacy. Members of the WG will be 
external to NIH to assure independent evaluation. WG members will be vetted for conflicts of 
interest and will be required to disclose any potential conflict that arises during the study. The 
WG will be composed of both United States and Costa Rican nationals. The WG will be broadly 
charged with advising the NCI Principal Investigator and DCEG Scientific Director on scientific 
and policy issues that arise during the study. 
The WG will meet yearly during the vaccination phase of the trial and as required during the 
follow-up phase. After each review, members of the WG will summarize their findings and 
provide recommendations and will report them to the NCI Principal Investigator, the DCEG 
Scientific Director, and the NCI Board of Scientific Councilors (BSC). A WG charter document will 
detail the charge to the committee, membership, implementation and reporting plans. 
Regulatory Body Review 
NCI presented the trial concept to the EMA Vaccine Working Party on June 8, 2018 (London, 
England for preliminary assessment). The concept was favorably received, and investigators 
were encouraged to proceed and formally seek feedback from the EMA CHMP, along with GSK, 
the marketing authorization holder of Cervarix. The goal of engaging with EMA is to achieve a 
one dose label change for Cervarix, if the PRIMAVERA results meet the criteria for non-
inferiority.  
DATA SHARING PLAN 
Sharing research data supports the mission of the National Institutes of Health (NIH) and is 
essential to facilitate the translation of research results into knowledge, products, and 
procedures that improve human health. Whenever possible, data collected and/or generated 
from this study will be made available for secondary research purposes and will be made 
available no later than at the time of publication. Note that no genetic analyses will be 
conducted or made available. 

46 
 
Provisions will be taken to protect the subjects’ privacy and the confidentiality of the data, 
including but not limited to, de-identification of subject-level data and limiting secondary 
research use to what is deemed appropriate in the protocol and informed consent and/or 
assent forms signed by research participants and their guardians. If there is a question about 
whether a proposed analysis is authorized within the signed IC, the Working Group, and/or 
Costa Rican IRB and NCI ERP may be consulted. 
When data are shared outside of the NIH for collaborative and non-collaborative research, all 
data will be anonymized; transfers will be recorded and appropriate approvals will be obtained 
and documented for human subject and Privacy Act compliance. At minimum written 
documentation between the NIH and the recipient of the data must be made to memorialize 
what is being shared and under what conditions. This may be accomplished via Data Transfer 
Agreements (DTAs) between the NCI and the recipient institution(s). If data are to be shared via 
a publicly accessible research repository, additional documentation and application 
requirements may apply. 
The study investigators reserve the right to require payment to its contractors to cover their 
costs to produce data sets and accompanying documentation. 
 
 

47 
 
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NCI IRB/ERP AMENDMENTS AND APPROVAL DATES 
 
Approved 
Description of Amendment 
13 Feb 2019 
Versions 1.1 and 1.2 (submitted to the NCI IRB as one amendment): 
Clarifications and revisions made to the protocol in response to review 
comments and changes requested by the Costa Rican IRB, as well as 
some issues identified by the investigators. Some of changes involved 
corresponding modifications to the assent and consent forms, as well as 
new materials, namely the consent video scripts. 
27 March 2019 
Version 1.3: Revisions to the methods, analysis plan, and procedures 
related to a new follow-up visit one month post-vaccination to collect 
blood from the Cervarix group (girls ages 9-14). This change was 
recommended by the CVT Working Group as a means to bridge the 9-10 
year olds to the 11-14 year olds in the event that initiation of HPV 
vaccination in fourth graders, as part of the National Immunization 
Program, requires exclusion of the 9-10 year olds from the primary 
analysis. Corresponding modifications also were made to the forms and 
videos for assent and parental consent, and the information form. The 
new re-assent form for participants who turn 12 during follow-up was 
also included, as was the new invitation PowerPoint for recruitment. 
22 May 2019 
Version 1.4: Revisions to the objectives, overview, and analysis plan 
requested by the EMA, a clarification to the content regarding the field 
response to the national vaccine program, and a change in the exclusion 
criteria to remove latex, and an increase in the accrual ceiling to permit 
the enrollment of up to 100 additional participants per study group.  
7 July 2022 
Version 1.5: Revisions to the analysis plan to incorporate evolving 
knowledge about the impact of the national vaccine program, and 
changes to reflect that NCI ethical oversight will be conducted by an 
ethics review panel, no longer the NCI IRB. 
3 March 2023 
Version 1.6: Revisions to the analysis plan to add HPV6/11 testing of 
selected participants and move SEAP testing to 36-month visit.