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months post partum and no longer breastfeeding. No screening for HPV DNA or antibodies was performed before enrollment/vaccination. The trial was reviewed and approved by human subjects review committees of the National Cancer Institute in the United States and INCIENSA (Insti- tuto Costarriciense de Investigacion y Ensenanza en Nutricion y Salud) in Costa Rica.

Data and Specimen Collection Prior to Randomization/Vaccination

A risk factor questionnaire was admin- istered to all eligible participants who consented to participation. A pelvic examination was performed on all sexually experienced women, at which time exfoliated cells were collected in Preservcyt liquid medium (Cytyc Corp, Marlborough, Massachusetts) for Thinprep (Cytec Corp) cytologic evaluation and for HPV DNA, Chla- mydia trachomatis, and Neisseria gon- orrhoeae testing. Blood specimens were also collected from all partici- pants prior to randomization and vac- cination; serum samples obtained from these specimens were used for HPV antibody testing.


Women in our trial were randomized at the site in a blinded fashion to re- ceive either the HPV-16/18 VLP vac- cine formulated with the AS04 adju- vant system or a control hepatitis A vaccine consisting of inactivated viral antigen formulated with alum (Havrix, GSK Biologicals). Study and control vaccines were assigned random vac- cine identification numbers at the time of labeling by the manufacturer. Study personnel at the Costa Rican study site randomized participants by assigning each eligible participant to the next available sequential vaccine identifica- tion number. The protocol called for a dose of vaccine at each of 3 study vis- its: at enrollment, 1 month following the initial dose (allowable range, 21- 120 days), and 6 months following the initial dose (allowable range, 121-300 days).

At the 6-month clinic visit, all sexu- ally experienced women were in- structed to self-collect a cervicovagi- nal specimen using a Dacron swab. Exfoliated cells from this collection were stored in Preservcyt solution and used for HPV DNA testing. Following this self-administered collection, women who at study entry had evi- dence of either atypical squamous cells of uncertain significance that were posi- tive for HPV by a molecular hybridiza- tion assay using chemiluminescence with HPV RNA probes (the Hybrid Cap- ture 2 [HC2] test; Digene Corp, Silver Spring, Maryland) or a low-grade squa- mous intraepithelial lesion had a pel- vic examination performed. At the time of this pelvic examination, a cervical specimen was also collected by a clini- cian and stored in Preservcyt solution.


Following the initial vaccination phase, all participants were asked to attend one of the study clinics approximately 1 year after enrollment (12-month visit). At the time of this annual screening visit, a pelvic examination was performed (on sexually active women) and exfoli- ated cells were collected by a clinician for cytologic evaluation and HPV DNA testing in a manner similar to that de- scribed for enrollment. Additional fol- low-up of participants beyond the 12- month period is ongoing.

Safety Monitoring

Participants remained at the clinic for 30 to 60 minutes following each vac- cination. Reactogenicity and adverse event experiences were collected from all participants during this period and in the week following each vaccina- tion via home visits for a 10% ran- domly selected sample of participants. Adverse event and pregnancy informa- tion was also actively collected from all participants at each of the follow-up clinic visits. In addition, a toll-free num- ber is continuously staffed by clinical personnel, and participants are in- structed to call this number between visits if they experience any medical event.

A data and safety monitoring board (DSMB) established by the National Cancer Institute to oversee the trial meets on a regular basis to evaluate safety data in closed session (date of most recent meeting: June 19, 2007). The DSMB has recommended trial continuation. The trial statistician (S.W.) participates in the closed- session review of summary tables of adverse events by group but is not involved in the formulation of the final DSMB recommendations. Since the ongoing main trial is still blinded, other investigators and site personnel have no access to safety data by treat- ment group; therefore, no safety data are presented herein. Safety data and the main prophylactic efficacy results will be reported once the final analysis for the main trial is begun.

HPV DNA Testing

Human papillomavirus DNA testing was performed on enrollment (prevac- cination) specimens using a 2-mL ali- quot of exfoliated cells stored in Pre- servcyt solution. Testing was performed using probe B (designed to detect 13 on- cogenic HPV types including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) per manufacturer’s instruc- tions in a laboratory located at the Uni- versity of Costa Rica in San Jose.20

Broad-spectrum polymerase chain reaction (PCR)–based HPV DNA test- ing was performed at Delft Diagnostics Laboratory (Delft, the Netherlands) using a previously described proce- dure based on amplification using the SPF10 primers and a DNA enzyme immunoassay detection of amplimers and, if positive, followed by typing using the line probe assay (LiPA) line blot detection system (Inno-LiPA HPV genotyping assay SPF10 system, ver- sion 1, Labo Bio-medical Products, Rijswijk, the Netherlands).21-23 The LiPA assay detects 25 HPV genotypes (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74). Testing was performed on a 0.5-mL aliquot of Preservcyt solution removed from the cytologic specimen prior to slide

©2007 American Medical Association. All rights reserved.

(Reprinted) JAMA, August 15, 2007—Vol 298, No. 7


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