Women included in the present evaluation are participants in a randomized clinical trial initially designed to evaluate the efficacy of a bivalent HPV vaccine against persistent type-specific HPV16/18 infection and associated precancerous lesions at the cervix (
15,
16). The study enrolled women residing in Guanacaste and selected areas of Puntarenas, Costa Rica, identified via a census, between 2004-2005. Main eligibility requirements were: age 18-25 years, planned residence in the area for the 6 months following enrollment, in good general health, neither pregnant nor breastfeeding, and willing to provide written informed consent. Women were excluded if they had preexisting medical conditions that required chronic treatment or caused immunosuppression, had a history of hepatitis A or previous vaccination against it, or were unwilling to use contraception during the vaccination period. 7,466 women were enrolled; they represented 59.1% of eligible and 30.5% of all (eligible plus ineligible) women from the census (
15). The trial was approved by the human subjects review committees of the United States (US) National Cancer Institute and INCIENSA (Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud) in Costa Rica.
At the enrollment visit (following informed consent and risk-factor interview), a pelvic examination was performed on sexually experienced women, exfoliated cervical cells were collected in PreservCyt medium (Cytyc Corp, now Hologic, Marlborough, Massachusetts) for Thinprep (Cytyc Corp) cytologic evaluation and HPV DNA testing, and blood was collected for HPV16/18 serology. Next, women were randomized in a double-blinded fashion to receive the HPV vaccine (
Cervarix, GlaxoSmithKline Biologicals) or a “Control” hepatitis A vaccine (a modified preparation of Havrix, GSK Biologicals). Both vaccines were formulated in three 0.5 ml doses with identical packaging. HPV and control vaccines were assigned random vaccine 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 identification number. The protocol called for a dose of vaccine at each of 3 study visits: 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). Women not attending their visits in the allowable range for the 2
nd dose (21-120 days after enrollment) remained eligible for the final dose; women who missed the window for the final dose (121-300 days after enrollment) did not receive that dose (
15).
At annual follow-up visits, clinicians collected from sexually active women exfoliated cervical cells (same method as above) for cytologic evaluation and HPV DNA testing Women with low-grade cytologic abnormalities were evaluated every six-months until 3 consecutive normal cytologic results returned them to yearly follow-up. Women with cervical high-grade disease or persistent low-grade abnormalities were referred to cervical colposcopy for evaluation and treatment if needed.
Sampling of the anus was introduced at the four-year study visit, the final blinded study visit of the trial. At this study visit, a questionnaire was administered that included questions on anal sexual behaviors; while some under-reporting of stigmatized behaviors such as anal sex is expected, we don't anticipate differential reporting by vaccination status that would bias our efficacy estimates.
The anal specimen was collected prior to the pelvic exam among sexually active women (defined by vaginal intercourse) by inserting a dry swab 3-4 cm into the anal canal, rotating one time, and then removing the swab while rotation continued using gentle pressure against the wall of the anal canal. The swab was placed in 1ml of PreservCyt and frozen immediately in liquid nitrogen. While the predictive value of one-time detection of anal HPV16 or 18 likely for anal precancer or cancer is likely very low, and there are not standard clinical recommendations for need of follow-up in such cases, a subset of women with anal HPV16/18 infection will be monitored during the long-term follow-up phase of the trial. Most women will be followed up to 10 years since initial vaccination; as part of this effort, long-term type-specific anal HPV persistence and related disease will be monitored, and clinical management will be provided if necessary.
Anal and cervical samples were tested for HPV DNA using the SPF
10 PCR primer system and a DNA enzyme immunoassay detection of amplimers (DEIA; DDL Diagnostic Laboratory [Voorburg, the Netherlands]); if positive, genotyping was conducted using the line probe assay (LiPA
25) (SPF
10PCR/LiPA
25 HPV genotyping assay system, version 1, Labo Bio-medical Products, Rijswijk, the Netherlands) (
17). LiPA
25 detects 25 HPV genotypes, including carcinogenic (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68/73) and non-carcinogenic (6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 70, and 74) types. To ensure that HPV16 and HPV18 infections were not missed, all positive specimens on SPF
10 PCR/DEIA that were negative for HPV16 or HPV18 by LiPA
25 were also tested using HPV16 or 18 type-specific primers (
18,
19).
Serum collected at enrollment was used to determine HPV16 and HPV18 serological status using a VLP-based direct ELISA, a standard measure that measures polyclonal antibodies (GlaxoSmithKline Biologicals, Rixensart, Belgium), as described previously (
20,
21). Antibody results were dichotomized using standard cutoff points calculated from antibody titer values 3 SDs above the geometric mean titers taken from a group of HPV-negative individuals (
20). Cut points were optical density of at least 8 EU/mL for anti-HPV16 and at least 7 EU/mL for anti-HPV18 (
20,
21).
Statistical Analysis
Characteristics between women who accepted and declined the anal specimen collection were compared using the chi-squared test for categorical variables. Among women who accepted anal specimen collection, general characteristics from both the enrollment and 4-year post-vaccination visits were compared between women in the HPV and control arms; this test shows that despite attrition over time, the two analysis groups remain roughly similar as per the randomization. Median follow-up time from enrollment was calculated in months overall and compared by arm using the Kruskal-Wallis test.
Prevalence of anal HPV 16 or 18 infections approximately four years after vaccination was the primary endpoint (defined as detection of either HPV16 or HPV18 or both at the four year study visit); cervical HPV 16 or 18 infections among the same women at the same time point were evaluated for comparison purposes.
The full analysis cohort (presented in the CONSORT ) included all women who had anal specimens collected and had HPV results available (“full cohort”); thus, no exclusions were based on HPV DNA positivity, HPV serostatus, or number of vaccine doses received, in this cohort. The “restricted cohort” included women from the full cohort with no evidence of prevalent cervical HPV16/18 infection or HPV16/18 antibodies prior to vaccination, who received three doses of the HPV or control vaccine. By restricting based on cervical infections which are correlated with concomitant anal infections (e.g.: in our population, type-specific HPV16 agreement at the anus and the cervix measured at the four-year study visit was 30.5% (47 out of 154) percent positive agreement and kappa of 0.44), we intended to remove women from this secondary analytic cohort who may have had past or prevalent anal HPV infection (
22), since a pre-vaccination anal specimen was not collected to allow for this exclusion based directly on anal HPV detection. Specifically, to compose the restricted cohort, women were excluded from the full cohort if they met any of the following criteria: 1) cervical HPV 16 or 18 DNA positive at enrollment (or missing results), 2) HPV 16 or 18 seropositive at enrollment (or missing results), 3) biopsied for possible cervical intraepithelial neoplasia (CIN) or treated by LEEP, following a positive screening test during the vaccination phase (e.g.: until the 6-month study visit, which could functionally occur 4-10 months after enrollment), or 4) recipients of fewer than three doses of either vaccine.
For each arm, the prevalences of anal and cervical HPV16/18 combined and separately measured four-years post-vaccination were expressed as the number of infected women per 100 women vaccinated (stratified by HPV vs. control vaccine); asymptotic confidence intervals (95%CI) were estimated except when cells had less than five events, in which case exact confidence limits were reported. The complement of the ratios of the prevalence for the HPV and control arms comprised the VE estimates. In this woman-level analysis, each woman could only contribute once to the numerator and denominator, even if multiple HPV types were detected. Exact confidence intervals (
23) for vaccine efficacy were calculated based on the binomial distribution of the number of events in the HPV arm among the total number of events in the HPV and control arms (
24). Anal and cervical VE estimates were calculated and compared by including an arm*anatomical site interaction variable in a GEE model (
25) and evaluating whether the beta coefficient for the interaction variable varied significantly from 0.
In our pre-specified plan, the main objective of our analysis was to evaluate VE against anal HPV16/18 infection four years following enrollment and administration of the first vaccine dose (regardless of vaccine type); cervical VE in the exact same cohorts was estimated as a comparator. Because of evidence for cross-protection in cervical VE studies (
10,
11), anal (and cervical) VE against a pre-specified composite endpoint of HPV31/33/45, and then individually by type, was evaluated. In the restricted cohort, women who were HPV31, 33, or 45 DNA positive at enrollment were excluded from each respective analysis; no serologic restrictions were made because serologic testing for these HPV types was not conducted. Anal and cervical VE against all other carcinogenic types (after removing HPV16/18/31/33/45) was evaluated in the full cohort.
HPV16/18 VE was estimated by self-reported anal sex (yes/no) assessed by questionnaire at the study visit when anal specimen collection occurred, in the full and restricted cohorts.
This trial is registered at clinicaltrials.gov, identifier: NCT00128661.
Role of the Funding Source The Costa Rica HPV Vaccine Trial is a long-standing collaboration between investigators in Costa Rica and the NCI. The trial is sponsored and funded by the NCI (contract N01-CP-11005), with funding support from the National Institutes of Health Office of Research on Women's Health, and conducted with support from the Ministry of Health of Costa Rica. Vaccine was provided for our trial by GlaxoSmithKline Biologicals (GSK), under a Clinical Trials Agreement with the NCI. GSK also provided support for aspects of the trial associated with regulatory submission needs of the company under FDA BB-IND 7920. Drs Schiller and Lowy report that they are named inventors on US government–owned HPV vaccine patents that are licensed to GSK and Merck and for which the NCI receives licensing fees. Drs Schiller and Lowy are entitled to limited royalties as specified by federal law. No other financial disclosures were reported. The NCI and Costa Rica investigators are responsible for the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation of the manuscript. The NCI and Costa Rica investigators make final editorial decisions on this and subsequent publications; GSK has the right to review and comment.
At the time of this analysis, fieldwork was on-going and individual information remained blinded. Thus, analyses were conducted by an external group, Information Management Systems (by Sabrina Chen; Rockville, MD), under the direction of the investigators who remained masked to individual random assignments. SAS 9.2 TS2M3 was used for analysis.
The publisher's final edited version of this article is available at 
