Study Population

From August 2012 through June 2015, we enrolled and followed healthy, sexually active, nonpregnant, HIV-1–seronegative women between the ages of 18 and 45 years at 15 research sites in Malawi, South Africa, Uganda, and Zimbabwe (Tables S1 and S2 in the Supplementary Appendix, available with the full text of this article at NEJM.org).16 The primary objectives were to determine the efficacy and safety of the dapivirine vaginal ring as compared with a placebo ring; after insertion, the ring is used for 4 weeks and then replaced with another. Community members from each site provided input into trial design and conduct. The trial protocol, which is available at NEJM.org, was approved by the ethics review committee at each site (Table S3 in the Supplementary Appendix). All participants provided written informed consent.

Study Procedures

At enrollment, women were assigned in a 1:1 ratio, with the use of fixed-size block randomization, stratified according to site, to receive either a silicone elastomer vaginal matrix ring containing 25 mg of dapivirine or a placebo vaginal ring. Both the dapivirine and placebo rings were manufactured by QPharma under contract with the International Partnership for Microbicides. The dapivirine and placebo rings were indistinguishable, and with the exception of staff members at the central statistical and data management center, investigators and participants were unaware of the randomization assignments until completion of the trial. Women were taught how to insert and remove the vaginal ring and counseled to wear it for the entire month.

Women returned for monthly follow-up visits, which included HIV-1 serologic testing, safety monitoring, and individualized adherence counseling (Table S4 in the Supplementary Appendix). At each visit, a new ring was provided, and the ring that had been used during the previous month was collected. Women were tested monthly for pregnancy, and the study ring was withheld from women who became pregnant; they resumed use of the study ring when no longer pregnant or lactating. All participants received a package of HIV-1 prevention services, including counseling with respect to HIV-1 risk reduction, partner HIV-1 testing, treatment of sexually transmitted infections in participants and partners, and free condoms. (Details regarding the trial design are provided in the Supplementary Appendix.)

Objective Assessment of Adherence

Plasma samples that were collected quarterly were tested for the presence of dapivirine with the use of a validated ultra-performance liquid chromatography–tandem mass spectrometry assay (Clinical Pharmacology Analytical Laboratory), with a lower limit of quantification of 20 pg per milliliter.17 To aid in distinguishing cases in which the ring was removed during the month and then reinserted before a clinic visit, the detection of a plasma dapivirine level of more than 95 pg per milliliter (a level nearly always achieved within 8 hours of continuous use) was used to define adherence.13-14 While the trial was ongoing, plasma samples were assayed and results were reviewed by the trial leaders. To preserve blinding, samples from both the dapivirine group and the placebo group were tested, and results were summarized only as the percentage of samples with dapivirine detected, overall and for each site. After the first year of the trial, testing for residual dapivirine in used rings was initiated with the use of acetone extraction and high-pressure liquid chromatography (Parexel). Women were defined as being adherent if the returned ring contained less than 23.5 mg of dapivirine (i.e., with >1.5 mg released).13-14

Primary End Points

The primary efficacy end point was HIV-1 infection, identified with the use of a standard seroconversion algorithm (Fig. S1 in the Supplementary Appendix). The study ring was temporarily withheld while confirmatory testing was pending and was permanently discontinued if testing confirmed HIV-1 acquisition. Archived plasma samples from visits before seroconversion were tested for HIV-1 RNA on polymerase-chain-reaction (PCR) assay, and participants with detectable HIV-1 RNA at the time of enrollment were excluded from the primary analysis. Participants completed a final study visit 4 weeks after the last product-use visit to assess for delayed HIV-1 seroconversion, and women who tested positive for HIV-1 at that visit and who had detectable HIV-1 RNA at the last product-use visit were included in the primary analyses because HIV-1 infection had occurred during the product-use period.

The primary safety end point was a composite of any serious adverse event, any grade 3 or 4 adverse event, and any grade 2 adverse event that was assessed by the trial clinicians as being related to dapivirine.

Study Oversight

The National Institutes of Health funded the trial. The authors designed the trial, gathered and analyzed the data, prepared the manuscript, and were responsible for the decision to submit the manuscript for publication. The International Partnership for Microbicides supplied the vaginal rings, was the regulatory sponsor, and participated in the design of the trial, the interpretation of the results, and the preparation of the manuscript. The ring manufacturer, QPharma, had no role in the design or implementation of the trial. The authors vouch for the accuracy and completeness of the data and analyses.

Statistical Analysis

The trial was designed with power of 90% to detect a risk of HIV-1 infection that was 60% lower in the dapivirine group than in the placebo group, with a one-sided alpha level of 0.025. Like other trials of new HIV-1 prevention interventions,6,7,18 this trial was powered so that the lower boundary of the 95% confidence interval would exclude a 25% reduction in risk, with the primary analysis comparison for the trial planned against a standard null of 0% (Table S5 in the Supplementary Appendix). Under these assumptions, a minimum of 120 HIV-1 acquisition events would be required to achieve the statistical power posited in the design of the trial. An end-point–driven design was used, and the trial continued until the target number of HIV-1 end points had been accrued and all participants had been followed for a minimum of 12 months, in accordance with regulatory guidance regarding compilation of safety information for new HIV-1 prevention strategies.19

An annual incidence of HIV-1 infection of 3.9% in the placebo group was assumed, and a sample size of 3476 women was planned. After the trial started, another HIV-1 prevention trial that was conducted at several sites in our trial showed an HIV-1 incidence of more than 5% per year.3 As a result, in October 2013, the sample size for this trial was recalculated to approximately 2600 women, and the statistical analysis plan was modified accordingly. At the same time, the analysis plan was further modified for a fully powered analysis that would exclude all data from 2 of the 15 sites, since these 2 sites had shown lower-than-anticipated participant retention and lower product adherence (with adherence levels of <50%, according to measurement of plasma dapivirine levels) than at the other sites. Further enrollment at the 2 sites was discontinued, but enrolled participants were permitted to continue in follow-up. The sample-size recalculation and plan to exclude data from the 2 sites were approved by the independent data and safety monitoring board and reviewed by regulatory agencies. Interim statistical monitoring, which was performed on the basis of data from all 15 sites, used the Lan–DeMets spending approach to adjust the O’Brien–Fleming sequential monitoring boundaries.20,21

The primary analysis of HIV-1 protection was performed according to the intention-to-treat principle with the use of Cox regression, stratified according to site, to estimate the relative rates of time until HIV-1 acquisition. Two analyses were defined: one included data from all 15 sites and a second excluded data from the 2 sites at which enrollment had been discontinued early (i.e., 13 sites were included). Prespecified subgroup analyses were planned. When it was determined that age was significantly related to the efficacy of HIV-1 protection, a post hoc analysis was designed to characterize more fully that relationship by dividing the population into age-categorized thirds containing approximately equal numbers of participants with HIV-1 infection, thus balancing the statistical power for the exploratory subgroups. All analyses were conducted with the use of SAS software, version 9.4 (SAS Institute), and R software, version 2.15.1 (R Project for Statistical Computing). A P value of less than 0.05 was considered to indicate statistical significance, and all P values are two-sided.