Safety and Persistent Immunogenicity of a Quadrivalent Human Papillomavirus Types 6, 11, 16, 18 L1 Virus-Like Particle Vaccine in Preadolescents and Adolescents: A Randomized Controlled Trial

Keith S. Reisinger, MD, MPH; Stan L. Block, MD; Eduardo Lazcano-Ponce, MD; Rudiwilai Samakoses, MD; Mark T. Esser, PhD; Joanne Erick, BA; Derek Puchalski, BS; Katherine E. D. Giacoletti, MStat; Heather L. Sings, PhD; Suzanne Lukac, BS; Frances B. Alvarez, RN, BSN; Eliav Barr, MD


Pediatr Infect Dis J. 2007;26(3):201-209. 

In This Article


Between October 2003 and March 2004, 1781 healthy, sexually naive boys and girls 9 to 15 years of age were enrolled at 47 study sites located in 10 countries in North America, Latin America, Europe and Asia. Inclusion/exclusion criteria were similar to that described for a noninferiority immunogenicity bridging study.[26] An Institutional Review Board for each clinical site approved the study protocol. At enrollment, written consent was obtained from each participant and his/her legal guardian.

The quadrivalent HPV-6/11/16/18 L1 VLP vaccine (GARDASIL/SILGARD, Merck and Co., Inc., Whitehouse Station, NJ) has been described.[22] The placebo used in this study contained identical components to those in the vaccine, with the exception of HPV L1 VLPs and aluminum adjuvant, in a total carrier volume of 0.5 mL. Vaccine and placebo were visually distinguishable.

The trial (Merck protocol V501-018) was a randomized, double-blind (with sponsor blinding), placebo-controlled, multicenter study. Enrollment was stratified by age (2:1 ratio of 9- to 12-year-old subjects and 13- to 15-year-old subjects) and by gender (1:1). Randomization schedules were computer-generated using a blocking factor of 6. An interactive voice response system was used to allocate study subjects and to assign allocation numbers. Subjects were randomized in a 2:1 ratio within study centers to receive 3 intramuscular injections of either quadrivalent HPV vaccine or non-aluminum-containing placebo at day 1, month 2 and month 6. The deltoid muscle was the preferred site for intramuscular injection. Vaccine/placebo was administered using a 1.0-mL syringe with needle length of 1 to 2 inches (22-23 gauge).

As the vaccine and placebo used in this study were visually distinguishable, they were prepared and administered by unblinded study personnel not otherwise involved in the care and management of the study participants. The success of blinding was assessed by designated unblinded sponsor and study personnel. To ensure effective monitoring of adverse experiences, an independent safety monitor (not employed by the sponsor) was used. Otherwise, the subject and the investigator, study site personnel, and laboratory personnel conducting the clinical assays were blinded to vaccination group throughout the study. The sponsor's clinical, statistical and data management teams were blinded until the primary analysis at month 7.

A medical history and physical examination were conducted at day 1. If the participant was found to have a temperature of ≥100°F (oral) within 24 hours before an injection, the injection was postponed. For all female subjects, a pregnancy test (sensitive to 25 IU human chorionic gonadotropin) was performed prior to each injection.

Blood samples were obtained on day 1 prior to the first vaccination, month 7 and month 18. Serum specific neutralizing antibodies to HPV-6/11/16/18 were measured using a competitive Luminex immunoassay (cLIA), as described.[27] Seropositivity was defined as anti-HPV serum cLIA levels ≥20, 16, 20 and 24 mMU/mL for HPV-6, HPV-11, HPV-16 and HPV-18, respectively.[27] Seropositivity information was not available prior to the day 1 vaccination. However, if a subject was found to have anti-HPV levels above any serostatus cutoffs at day 1 (prevaccination), indicating prior exposure to one or more vaccine HPV-types, this result was communicated to the primary investigator who had enrolled the subject. After unblinding of the data, investigators were to communicate the finding to the subject and to the parent/legal guardian with appropriate counseling.

Participants were observed for at least 30 minutes after each vaccination for any immediate reaction. Temperatures were recorded orally for 5 days following each injection. All adverse experiences were collected daily by the parent/legal guardian on a vaccination report card for 14 days following each vaccination. Follow-up at months 2, 6, 7, 12 and 18 included an interview to assess general safety. In addition, at any time during the study, all deaths (regardless of cause) and serious adverse experiences that were considered by the investigator to be vaccine-related were to be reported. The relationship between adverse experiences and vaccine was reported by the investigator according to his/her best clinical judgment, based on exposure, time course, likely cause and probability with vaccine profile.

Safety. The primary safety hypothesis stated that a 3-dose regimen of quadrivalent HPV vaccine is generally well tolerated in adolescents and preadolescents. A detailed tolerability analysis was performed with emphasis on the following prespecified adverse experiences: vaccine-related adverse experiences, injection-site adverse experiences (swelling/redness and pain/tenderness/soreness), systemic adverse experiences (muscle/joint pain, headaches, hives, rashes and diarrhea), severe adverse experiences, and fever. All subjects who received at least one injection and had follow-up data were included in the safety summaries. Adverse experiences were summarized descriptively as frequencies and percentages by vaccination group and type of adverse experience, by vaccination visit and across all vaccination visits. Elevated temperatures (≥100°F oral or oral equivalent) within 5 days following each vaccination were summarized in a similar manner. In addition, risk differences and associated 95% confidence intervals (CI) were computed comparing the vaccine and placebo groups across all vaccination visits with respect to adverse experiences with ≥1% incidence in either vaccination group. P values were computed only for those adverse experiences that were prompted for on the vaccination report card (elevated temperatures, injection-site pain, injection-site swelling, injection-site redness, muscle/joint pain, headaches, hives, rashes and diarrhea). Adverse experiences were also summarized separately for boys and girls (within each vaccination group) and by age group. No formal comparisons were made between boys and girls or age groups with respect to adverse experiences.

The secondary hypothesis of this study stated that the immune responses to quadrivalent HPV vaccine in preadolescent and adolescent boys are noninferior to the responses in preadolescent and adolescent girls, as measured by anti-HPV GMTs and seroconversion rates one month postdose 3 (month 7). Analyses of noninferiority were conducted based on HPV type-specific per-protocol populations. The per-protocol populations for HPV-6, HPV-11, HPV-16 and HPV-18 consisted of subjects who were seronegative to the relevant HPV type(s) at enrollment, received all 3 doses of vaccine or placebo within the protocol-specified time frames and did not violate the protocol.

The evaluation of noninferiority of boys to girls with respect to the percentage of subjects who seroconverted for each HPV type by month 7 used 4 one-sided tests of noninferiority (one corresponding to each HPV type) conducted at the 0.025 level, based on the methods of Miettinen and Nurminen.[28] To reject the null hypothesis for a given HPV type, the lower bound of the 95% CI on the difference in the percentages of seroconverters (boys minus girls) for that type had to be greater than -5 percentage points.

Noninferiority of boys to girls with respect to month 7 anti-HPV GMTs was tested using an analysis of variance (ANOVA) model. The natural log of the individual titers of the subjects in the quadrivalent HPV vaccine group was modeled as a function of gender, age at enrollment and geographic region, which were considered fixed effects. The analysis was performed using the mean squared error from the ANOVA model as an estimate of variance and a one-sided test for the similarity of 2 means was performed at the 0.025 level. The antilog of the estimated treatment difference in the ANOVA model, and the associated 95% CI, was computed. To reject the null hypothesis for a given HPV type, the lower bound of the 95% CI on the ratio of month 7 GMTs (boys/girls) for that type had to be greater than 0.5.

To declare the immune responses in boys noninferior to those in girls, the statistical criterion had to be met for each HPV type and for each endpoint (month 7 GMTs and seroconversion rates).

A secondary immunogenicity objective was to describe the persistence of immune response to the quadrivalent HPV vaccine. To address this objective, GMTs for each vaccine HPV type, with associated 95% CIs, were summarized at month 18, 1 year following completion of the primary vaccination regimen. In addition, percentages of per-protocol subjects who remained seropositive at month 18 were calculated.

The primary hypothesis in this study relates to the tolerability of the quadrivalent HPV vaccine. If no vaccine-related serious adverse experiences were observed among 1100 vaccinated subjects, this study provided 95% confidence (one-sided) that the true incidence was no greater than 0.27%. With at least 847, 847, 836 and 836 evaluable subjects in the per-protocol populations related to HPV-6, HPV-11, HPV-16 and HPV-18, respectively (based on predicted rates of attrition and baseline seropositivity rates), the study had >99% power to rule out a ≥2-fold difference in the ratio of GMTs (α = 0.025, 1-sided) for each vaccine HPV type and >99% power to rule out a ≥5-percentage-point difference in seroconversion rates between the 2 groups (α = 0.025, 1-sided) for each HPV type. If the 2 immunogenicity hypotheses were independent, the overall power of the study to declare noninferiority of immune responses in boys relative to girls was >99%.

The analyses presented here include all safety and immunogenicity data from visits that occurred on or before November 7, 2005. At this time point, the duration of participation was approximately 1.5 years.

The studies were designed by the sponsor (Merck and Co., Inc.) in collaboration with clinical site investigators. The sponsor collected the data, monitored the conduct of the study, performed the statistical analysis and coordinated the writing of the manuscript with all authors. Data were unblinded for statistical analyses after the databases were locked. The authors were actively involved in data analysis and interpretation and approved the final manuscript. All authors vouch for the veracity and completeness of the data and the data analyses.


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