Design and objectives of the study
The study described in this document is a community-based double-blind randomized controlled phase III trial of a virus-like particle (VLP) vaccine against HPV types 16 and 18 among healthy women 18–25 years old. The control vaccine is inactivated antigen of hepatitis A virus (three dose formulation used in Twinrix® that allowed blinding to be maintained).
The main objective of the trial is to investigate the efficacy of the vaccine to prevent cervical cancer precursors (defined as persistent HPV infection and/or high grade cervical intraepithelial neoplasia, CIN2 or CIN3).
We are particularly interested in evaluating the potential effectiveness of the vaccine at the population level, the impact of the HPV vaccine in relation to previous HPV exposure and age at vaccination, and will explore in detail efficacy against non-vaccine HPV types, duration of protection, characteristics of the immune response to the vaccine and potential impact of less than 3 doses.
We have recently reported on the lack of therapeutic efficacy among women who were HPV positive at enrollment into the study (21
). In addition, multiple tertiary objectives are envisioned, to address issues of relevance to HPV vaccination, to understand immunological responses to vaccination and to further investigate the natural history of HPV and HPV-associated outcomes.
The trial included eligible women 18 to 25 years of age residing in Guanacaste Province, Costa Rica or selected areas of Puntarenas Province. Consenting women were randomized to receive the candidate vaccine (40 µg of L1 protein VLPs of HPV 16 and 18 formulated with ASO4 adjuvant, consisting of alum and monophosphoryl lipid A) or the vaccine against hepatitis A consisting of 720 ELISA Units of inactivated viral antigen with Alum, formulated in 0.5 ml doses for this study. Randomization was carried out in a 1:1 ratio (). The vaccination schedule consisted of 3 doses, one at the enrollment visit, one a month later and one 6 months after recruitment.
Design of the HPV 16/18 vaccine trial in Guanacaste
After receiving the vaccines, women are being followed once a year for at least 4 years, as long as they have normal cytology. If they have minor HPV-related cytological abnormalities they are transferred to a 6 month schedule and if the lesion persists they are referred to colposcopy. Also, if they have high grade cytology at any time during the study, they are referred to colposcopy for diagnosis and treatment (see below).
Organization of the study
During the vaccination phase of the study, 7 study clinics were established with appropriate facilities to conduct the trial procedures. The clinics were located in Liberia, Cañas, La Cruz, Nicoya, Tilarán, Santa Cruz and Puntarenas. The staff at each clinic included a physician, a microbiologist who functioned as team coordinator, a nurse and a nurse aide, an assistant team coordinator, a supervisor, a driver, an interviewer and a receptionist. The clinics were equipped to provide emergency care and the staff was extensively trained for medical emergencies, particularly anaphylactic reactions.
The headquarters in Liberia coordinated appointments and supervised the field work, using a data management system developed specifically to assist the process, and also housed the repository for vaccines and specimens, document center, resident doctors, data entry, information technology and quality control. All participant records, vaccines and specimens were centralized at the Liberia headquarters and transported daily to and from the clinics in study vehicles. The cytology and histopathology laboratories were also in Liberia, although interpretation of cytology was carried out at the private laboratory of the study cytopathologist (MA). Similarly, interpretation of histology was done at the lab of our pathologist (DG). The processing of blood samples and the CBC at enrollment was contracted to a local laboratory (Trilab Sci, Liberia). HPV DNA, Chlamydia trachomatis (CT) DNA and Neisseria gonorrhea (GC) DNA detection by Hybrid Capture 2 (HC2) on the remaining PreservCyt after cytology slide preparation were performed at the University of Costa Rica (UCR) in San José. Heparinized blood samples were transported every day from study clinics to the UCR for cryopreservation of lymphocytes.
Selection of the population and invitation to participate
The study is being conducted in Guanacaste, Costa Rica, an area in the northwest part of the country with a population of 292,500 inhabitants (2005), where we have been conducting our studies for many years. We also invited women from selected towns in nearby Puntarenas (total population 399,500). These areas are traditionally rural and agricultural, but tourism-related activities are expanding rapidly. Cervical cancer incidence and mortality have been traditionally high (>20 per 100,000 women per year).
A complete census of women ages 12–22 was conducted specifically for this study between February and July 2000 (anticipating a subsequent start a few years later within the age range of 18–25), with an update of urban areas in 2005. During the census, all households in the selected areas were visited by study outreach workers, to obtain name, date of birth, cédula (national unique identification number) or other ID number, exact address, geographic location (cantón, district, censal segment (like a census tract) and house number), details of a contact person, telephone number and information on stability of residence for all resident women ages 12–22. A total of 68,662 households in Guanacaste and 22,294 in Puntarenas were visited. Intensive duplicate checks were carried out as an ongoing process throughout the study. Detailed maps were created for each of the censal segments, including location and number of each house to facilitate subsequent location of the women by the outreach workers.
All women in the census were randomly assigned a study-specific individual personal identification number (PID) in the database, and when the study initiated, attempts were made to visit all women in the census who were at that time 18 to 25 years old, to invite them to participate in the study.
Outreach workers visited potentially eligible women at home to give them a personalized letter with an appointment to the nearest of our clinics and an informational brochure (see web page www.proyectoguanacaste.org
for educational materials). Women who appeared eligible and expressed an interest in participating were given a copy of the informed consent document to read and discuss with their families before the appointment. Women were offered transportation in the study vehicles or reimbursement of travel expenses, but they were not paid for participating in the study.
Eligibility criteria and informed consent
The day of the visit, the receptionist at the clinic verified the identity of the potential participant, updated personal information, showed her a video (see web page) explaining the design and procedures of the study and administered an initial eligibility screener. Women who appeared potentially eligible had an extensive discussion of the informed consent document with a trained interviewer, with nurses and doctors always available to answer questions by the participants. The consent form, approved by the Costa Rica and NCI IRBs (see below), included details on HPV, the study, the meaning of participating, risks and benefits associated with participation, use of biological specimens and confidentiality among other topics (available at www.proyectoguanacaste.org
). Women who decided not to participate or who were deemed ineligible for any reason were offered a physical exam and a Pap smear with a follow-up colposcopic evaluation and treatment if needed at no cost to them.
After signature of the informed consent, an interview on risk factors was administered by trained interviewers, with responses entered directly into pre-coded data fields on a computer screen. The questionnaire elicited information on years of education, marital status, income, household facilities, menstrual history, sexual, reproductive and contraceptive history, smoking and family history of cancer. Among lifetime sexually monogamous women, additional questions were asked about their sexual partner, including age, education, circumcision, sexual history and smoking (interview available upon request).
The process continued with a complete medical history and physical exam. A urine sample was collected to conduct a pregnancy test. After this was completed, study doctors assessed final eligibility for the vaccine trial. To be eligible for the study, women had to be non-pregnant and, if they reported having started sexual activity, had to be using some form of contraception at least one month before the application of the vaccine and be willing to use it until 2 months after the last dose. Acceptable methods included abstinence, condoms, hormonal contraceptives, IUDs and female surgical sterilization. Additional eligibility criteria included being in good general health as ascertained by the study clinicians. Exclusion criteria comprised chronic diseases, history of severe allergic reactions to vaccines, history of hepatitis A or previous vaccination against it, history of chronic administration of immunosuppressive drugs or immunosuppressive conditions, hysterectomy, use of other investigational products in the past 30 days, previous administration of the adjuvant in the candidate vaccine (ASO4), previous HPV vaccination, allergy to 2 phenoxyethanol or neomycin (components of the vaccine), or latex hypersensitivity (component of syringe).
Participation was deferred if a woman was sexually active and not using but willing to use contraception as per the protocol, was pregnant or less than 3 months post partum, was lactating, had an acute condition expected to resolve soon or had recent administration of a vaccine or immunoglobulin.
Clinical procedures and specimen collection
As mentioned above, after the interview a medical history and physical exam were carried out by the study doctors. After eligibility determination, a pelvic exam was performed on sexually experienced women. During the pelvic exam, cervical secretions were collected with polyvinyl acetate-based Mero-cell sponges (Medtronic Solan, Jacksonville, FL, USA), by placing one sponge on the cervical os gently for 30 seconds; and then a second one for the same length of time. The sponges were placed into empty 10 ml tubes and frozen in the vapor phase of liquid nitrogen immediately.
Exfoliated cells for cytology, HPV DNA, CT DNA, GC DNA and other testing were collected with a Cervex brush by firmly rotating the brush 5 times 360° around the cervical os. In women whose cervix exhibited extensive ectopy, the cervex brushing was also used on the ectocervix to insure sampling of the squamo-columnar junction. The brush was vigorously rinsed in liquid transport medium (PreservCyt) and stored in coolers at about 20° Celsius. An additional Dacron swab was used to obtain more cells for experimental HPV and other testing, by rotating it 360° in the cervical os, and placing the swab in UCM (universal collection medium) that allows preservation of RNA (Digene Corporation, Gaithersburg, MD (now Qiagen)). The swab, in medium, was placed immediately into a portable liquid nitrogen transport tank to optimize the quality of RNA.
The PreservCyt vial was processed as follows: first, two 0.5 ml aliquots were extracted following PCR-safe procedures for type-specific HPV detection. Then, a cytology slide was prepared with a ThinPrep 2000 processor, to obtain a thin-layer, liquid-based cytology preparation that was stained with a modified Pap stain. The remainder of enrollment specimens was sent to the HPV lab at the University of Costa Rica to test for HPV, CT and GC by hybrid capture 2 (HC2, Digene –Qiagen). The hybrid capture HPV results were used to triage women with ASC-US (see below) and CT and GC testing was done as a service for participants, who received treatment for themselves and their partners as appropriate. An analysis of the epidemiologic determinants of CT infection, found at baseline in 14.2% of participants, has been published (22
). During follow-up, HPV testing is restricted to women with ASC-US for triage, and CT and GC testing is done on women who initiate sexual activity or post-treatment to verify treatment efficacy.
Blood was collected at every visit regardless of previous sexual activity. At the enrollment visit, blood was collected to obtain serum and plasma. The specimen with citrate as anticoagulant was aliquotted to obtain buffy coats and plasma, and ascorbic acid and metaphosphoric acid buffers were added to preserve folic acid (whole blood) and vitamin C, respectively. Also, as a benefit for the participants, a specimen for a CBC was collected. At subsequent visits, only blood for serum is collected. Among 10% of all women and women developing HPV related cervical disease, an additional 40 ml of blood were collected in heparinized tubes, and sent to a laboratory at the University of Costa Rica for cryopreservation of lymphocytes using the ficoll-hypaque gradient method for studies of cell-mediated immunity.
Randomization (1:1) occurred in a masked fashion at the field site at the time the participant received her first vaccine dose. To allow for this, a range of vaccine ID numbers were randomly assigned to two groups by the Data Management Center. One group of random vaccine ID numbers was used at the manufacturing plant located in Belgium to label the HPV-16/18 bivalent vaccine. The other was used to label the control Hepatitis A vaccine at the same facility. Vaccine syringes for doses one, two and three were labeled in this manner. Doses two and three vaccine IDs were individually linked to dose one vaccine IDs using a vaccine ID numbering system that utilized an alphanumeric root ID number (e.g., VX12345) followed by a three digit sequence number that identified the dose number (i.e., 001, 002, and 003). Following the labeling process, vaccine vials from the two groups were combined in sequential order. This was done separately for each of the three doses. Vaccines were then shipped from the manufacturing plant to Costa Rica. In Costa Rica, first dose vaccine vials were dispensed in sequential order. Once a woman had been linked to a dose one vaccine ID, her vaccine ID for the remaining doses was fixed to the same root vaccine ID number (e.g., if a woman received vaccine VX12345-001 for dose 1, she was linked to VX12345-002 and VX12345-003 for doses two and three). This ensured that the same material type was administered to each woman at all three vaccination visits. To allow for replacement of vaccine vials that were damaged or otherwise unusable, additional doses were always available at the site. In cases where replacement was necessary, a web-based system developed and maintained by the Data Management Center was accessed by authorized personnel with valid logon and password information and a request made for a replacement vaccine. The web-based system provided the site with a replacement vaccine ID number without providing treatment arm information to ensure masking was maintained.
After the exams and specimen collection, women received their first vaccine most of the times in the non-dominant deltoid muscle, with a 1-inch syringe needle in most instances but a 1.5-inch syringe needle for large women. Extensive verification procedures were in place to make sure the women received the correct vaccine (i.e., the next available for the first dose and the vaccine with the same ID number as the first one for booster doses).
The first vaccination was given at the enrollment visit, and the second and third doses were targeted at one and six months after the initial dose was received. Two types of windows (allowable periods to be eligible for each study visit) were defined for each booster: the desirable window that complied with recommendations of the manufacturer and an allowable range that permitted vaccination outside the desirable window when necessary. The desirable window for the 1 month visit was from 21 days to 90 days after enrollment (first dose), and the allowable window was from 21 days to 120 days after enrollment. Similarly, for the 6 month visit, the desirable window was from 90 days to 210 days after the 1 month visit, and the allowable window was from 121 days to 300 days after enrollment. A woman who missed her allowable window moved to the next window and missed that vaccine dose.
During the 6-month visit, no pelvic exam was done unless the woman had an ASC-US positive for HPV DNA or LSIL interpretation of the enrollment cytology. However, all sexually active women were asked to self-collect a vaginal specimen for HPV testing. Detailed explanation of the procedure and a brochure were used to instruct the women on the collection of this specimen, which consisted of inserting a Dacron swab as high as possible into the vagina, trying to avoid touching the external genitalia, rotating it 5 times and placing it in an empty wide-mouth 50-ml collection cup. The nurse transferred the swab to a 10-ml vial with 3 ml of PreservCyt solution and placed it immediately in liquid nitrogen. Women who required a pelvic exam had both the clinician-collected specimen and the self-collected one (see results
A subset of women was invited to participate at a 7-month visit to obtain blood to investigate the maximum peak of immunogenicity. The target was to obtain 600 women for this component of the study
Monitoring of adverse events and pregnancies
All adverse events (any untoward medical condition occurring to a trial participant), independent of their possible relationship with vaccination are fully documented and followed through resolution using different mechanisms. The first assessment of reactogenicity was carried out immediately after each vaccination. Women were asked to stay in an observation room, initially for 30 minutes and, beginning in October 2005, for 60 minutes. After the observation period, women were asked about the occurrence of any reactogenicity symptoms, including fatigue, myalgia, arthralgia, headache, gastrointestinal symptoms, fever, malaise, difficulty breathing or rash. Ten percent of participants (those with a randomly assigned Participant ID [PID] ending in “2”) had a home visit scheduled by a study nurse 3–6 days after each vaccination. In addition, a toll-free number (800-doctora) with direct access to one of our resident doctors was available 24 hours a day, 7 days a week for reporting of adverse events or to answer questions by the participants. During subsequent visits, the participants are asked about hospital admissions or consultation with clinicians which, if reported, prompt the completion of an adverse event form. A rapid reporting system is in place to report serious adverse events within 24 hours to the IRBs and to a regulatory associate (Westat) for reports within regulatory timelines to NCI, the investigational new drug (IND) application holder (GSK) and the US Food and Drug Administration (FDA).
Pregnancies reported to any member of the study team are documented and reported to the NCI-contracted regulatory associates within 5 days of the study team learning about them. The regulatory associate communicates the information to NCI and the IND holder (GSK). All pregnant women are followed until resolution of their pregnancies and the outcome is documented, including characteristics of the delivery and the babies (duration of pregnancy, outcome, type of delivery, sex, weight, length, and Apgar score). Any abnormalities of a baby are reported as serious adverse events. In addition, all adverse events related to the pregnancy are reported. Statistics of non-serious adverse events are reported periodically to the corresponding authorities.
Follow-up is planned to continue for at least 4 years with yearly visits for most women. During the yearly visits, a pelvic exam is performed with collection of the same specimens as in the first visit, with the exception of citrated blood for plasma and whole blood for CBC. During follow-up, the 40-ml of blood for cryopreservation are collected in the subset of women with PID ending in “5” (immunogenicity subcohort) at visit 12 and 36, from women referred to 6 months follow-up (see below) and from women referred to colposcopy who require a biopsy or a LEEP. Women with LSIL or with ASC-US HPV positive are followed every 6 months until they have 3 consecutive normal cytologies (see below). The 4-year follow-up visit colposcopy referral algorithm (see below) will be modified using all available information (including HPV typing data) to assure the safety of the women before they are released from the study or transferred to a new follow-up schedule for optional long term follow-up (see below) under a separate protocol that has been developed.
Diagnosis and management of cytological abnormalities and colposcopic referral
Liquid-based cytologic preparations are made and stained at the Liberia laboratory following strict standard operating procedures and subject to extensive quality control methods. In particular, extensive measures to control humidity are essential to maintain the quality of the staining in tropical weather regions. Results are classified using the Bethesda system recording squamous and glandular changes.
Interpretation is carried out by a local laboratory with double reading by cytotechnologists and adjudication of all possibly abnormal interpretations by the cytopathologist (MA). The clinical management of the study participants relies on the Costa Rican cytopathology interpretation but, for quality control, all slides read as abnormal in Costa Rica (ASC or worse) and a 10% sample of the slides read as Negative (either totally Normal or Reactive Changes) in Costa Rica are re-screened and re-interpreted in the United States (led by cytotechnologist Claire Eklund and pathologist Martha Hutchinson). The 10% set of negatives selected for evaluation in the United States is randomly selected, without replacement and without regard to HPV test results. This sampling scheme was designed to result in approximately 50% of subjects with a negative cytology having an expert review over the course of the 4-year study (see results
Women with LSIL or HPV positive ASC-US are transferred from a yearly to a six-monthly follow-up schedule, until 3 consecutive normal cytologic results refer them back to yearly follow-up. A repeat LSIL or HPV positive ASC-US prompts referral to colposcopy, as does a single ASC-H or an HSIL+ at any time. The uncommon glandular abnormalities also lead to immediate referral to colposcopy. These guidelines are consistent with current American Society for Colposcopy and Cervical Pathology (ASCCP)-sponsored consensus guidelines for this age group (23
). Cytologic preparations interpreted as “Unsatisfactory” are treated as LSIL, referring women to semi-annual follow-up if previous interpretations were normal or to colposcopy if there was a previous LSIL or HPV positive ASC-US.
Colposcopic examinations are carried out by our expert colposcopist (JM), following strict diagnostic and treatment algorithms that involve biopsy of women with evident low-grade lesions and active surveillance with repeat colposcopy and cytology for women with apparently normal colposcopic impression. Women with histologic CIN2 or CIN3 lesions are treated with LEEP. Any woman with cancer would be referred for appropriate treatment at the local referral hospitals under the Social Security of Costa Rica. Women referred to colposcopy usually require several visits to complete their diagnostic workup; they do not attend regular study visits until released from colposcopy. After the last visit in the study, a modified algorithm for even more aggressive colposcopic referral, biopsy, and treatment will be established to assure safety of participants who might subsequently have less intensive surveillance than the trial provides.
Specimen handling, data management and quality control
Detailed standard procedures were developed for the proper labeling, transportation, processing and shipment of specimens, with particular emphasis on maintenance and monitoring of the cold chain. The vaccine and biospecimen repository operated in Liberia and the tens of thousands of specimens were tracked using the NCI biospecimen inventory system (BSI). Large vapor phase liquid nitrogen tanks were used for storage, small dry shippers for transportation from the clinics and large dry shippers for transportation to laboratories or repositories outside Costa Rica.
A data-management system was developed in collaboration with Information Management systems (IMS) and our computer experts. All data on case report forms were double-keyed and extensive data cleaning was carried out in real time by checking for inconsistencies. The data management system was designed to track the investigational product, handle appointments, permit data entry, and organize follow-up of adverse events and pregnancies. It also permitted a series of reports to assess the field effort. A strict safety and back-up protocol was established to assure integrity of the data, which were transferred periodically to IMS in the US, where extensive computer edits were conducted and queries produced to resolve all apparent discrepancies.
An extensive quality-control process was established, including continued training of the staff, careful documentation and tracking of deviations from procedures. Monitors from the NCI contracted regulatory associate reviewed 100% of informed consents, eligibility criteria, all serious adverse events and pregnancies, selected items in all charts after data entry and a sample of approximately 20% of all charts. Two of the NCI investigators served as medical monitors (MS and DS). They received safety reports, assisted the investigators with the adjudication of difficult cases and served as liaison between the Investigational New Drug (IND) holder (GSK) and Costa Rica.
HC2™ testing was performed on enrollment (pre-vaccination) specimens using a 2ml aliquot of exfoliated cells stored in Preservcyt. Testing was performed using Probe B (designed to detect 13 oncogenic HPV types including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68; and with known cross-reactivity to another carcinogenic type, HPV66) as per manufacturer’s instructions in a laboratory located at the University of Costa Rica in San José, Costa Rica.
Broad spectrum PCR-based HPV DNA testing was performed at Delft Diagnostics Laboratory (DDL, Delft, the Netherlands) using a previously described procedure based on amplification using the SPF10 primers followed by typing using the LiPA line detection system (24
). In addition, to ensure that HPV-16 and HPV-18 infections were not missed, all specimens that screened positive for HPV DNA using SPF10 but that were negative for HPV-16 or HPV-18 by LiPA were tested for the presence of HPV-16 and HPV-18 DNA using type specific primers, as previously described (26
). One of the two 0.5 ml aliquots of exfoliated cells stored in Preservcyt was used for PCR-based testing. Both enrollment specimens (pre-vaccination) and specimens collected at the semi-annual and annual visits were tested by the PCR method. For all except the 6-month visit, clinician administered cervical specimens collected during the pelvic examination were used. For the 6-month visit, self-collected cervicovaginal specimens were used. In addition, for the subset of women who had a pelvic examination performed at the time of the 6-month visit, a clinician-administered cervical specimen collected during the pelvic examination was used for PCR-based HPV DNA testing.
The SPF10/LIPA system in our study has shown very good agreement both with Hybrid Capture 2 (28
) and with Linear Array (29
The primary IRB reviewing and following the study was the INCIENSA (Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud) IRB. In addition, the CONIS (Costa Rican National Council for Health Research), the NCI IRB, and the IRB of the University of Costa Rica approved the study. A data and safety monitoring board (DSMB) was in place with members from Costa Rica and the US to carry out periodic evaluations of safety. In addition, an external Working Group was established; the CR and US members had varied expertise to provide scientific advice to the NCI.
In this article we present a comparison of the demographic characteristics of women included in both study arms. Statisitcal significance of the difference between arms was assessed with the chi square test, which was also used to test the significance of the difference between the Costa Rican census and the population included in our study.
To assess agreement between different laboratory tests or concordance between laboratories in the US and Costa Rica we used linear weighted kappa statistic and McNemar’s tests