Participants and design
MDP301 was a phase 3, randomised, double-blind, parallel-group trial
. Full details of trial design, sample size, research sites, study populations, study conduct including the randomisation and masking, and data underpinning the sample size assumptions, have been reported.18
We did a social science substudy to assess accuracy of behavioural and adherence data, which is described in detail elsewhere.19,20
Participants were enrolled at 13 clinics, which were managed by six research centres, in Africa (three in South Africa, one in Tanzania, one in Uganda, and one in Zambia). Eligible women were 18 years or older (≥16 years in Tanzania and Uganda); did not have HIV-1 infection at screening and were willing to be tested for HIV-1 infection and receive the result; were willing to have regular speculum examinations and urinary pregnancy tests; were willing to use gel as instructed; were likely to be sexually active; were willing to receive health education about condoms; and were willing and able to give informed consent. Women were not eligible if they were unable or unwilling to provide a reliable method of contact; were likely to move out of the area within 12 months; were likely to have sex more than 14 times a week on a regular basis (a regulatory requirement was that no more than 60 applicators were to be dispensed at every 4 weekly visit); used spermicides regularly; were pregnant or within 6 weeks post partum; had a severe clinical or laboratory abnormality; needed referral for assessment of a suspicious cervical lesion; had received treatment to the cervix or other gynaecological procedure within 30 days of enrolment; were allergic to latex; or were participating or had participated in another clinical trial that was likely to affect the primary efficacy endpoint within 30 days before enrolment.
The protocol was approved by local and national ethics committees, in all participating countries and in the UK. Authorisation was obtained from the national regulatory authority in all participating countries and the US Food and Drug Administration. Participants indicated their consent by signature or witnessed thumbprint.
Randomisation and masking
Participants were randomly assigned in a 1:1:1 ratio to 2% PRO2000, 0·5% PRO2000, or placebo groups. Participants were randomly assigned on the basis of lists that were created with randomised permuted blocks of varying size, for each of the 13 clinics, by an independent statistician with a computerised random number generator, containing unique trial numbers matched to nine sets of study product codes. Site pharmacists dispensed gel in identical applicators on the basis of the trial number and the assigned study product codes on the clinic randomisation list. No other site personnel had access to the list. Success of masking was not formally addressed. At enrolment, women were assigned a unique trial number selected sequentially from the clinic trial register. Only statisticians responsible for preparation of the Independent Data Monitoring Committee reports and essential manufacturing and distribution staff had access to the list matching study product codes to gel.
Visits were scheduled every 4 weeks for 52 weeks (up to 104 weeks in Uganda to provide long-term safety data). Gel was dispensed in packs of ten prefilled single dose applicators (maximum of 60 applicators in ten packs per visit), after a negative urinary pregnancy test. Women were instructed to apply gel within an hour before sexual intercourse. They were counselled to use condoms during all sex acts and received unrestricted supplies of free condoms at the research clinics. Gel supply was interrupted if a participant had a positive pregnancy test and could be resumed after a negative pregnancy test. At every 4 week visit, women were asked about gel and condom use at the most recent sex act and returned used and unused applicators, which were were counted and recorded for assessments of adherence.
HIV-1 status was assessed at 12 weeks, 24 weeks, 40 weeks, and 52 weeks (up to 104 weeks in Uganda) and when gel supply was interrupted or discontinued because of a positive pregnancy test. A clinical interview and pelvic examination to report genital and non-genital adverse events were also done at these visits, at week 4, and if a pregnancy was diagnosed. Adverse events were defined by ICH guidelines and MedDRA coded. Solicited genital symptoms and signs were non-menstrual bleeding, genital sores or ulcers, genital discomfort (itching, burning, or dryness), and external or internal epithelial disruption, genital erythema, and genital oedema.
Routine haematology and biochemistry tests were done for the first 500 participants enrolled in centres in Durban and Johannesburg, South Africa, and all 840 participants in Uganda at screening and at 12 weeks, 24 weeks, and 52 weeks (and 104 weeks or at final visit in Uganda). Additionally, a plasma sample was obtained from these participants at the final visit for PRO2000 analysis.
To confirm HIV-1 status, serum was obtained up to 6 weeks before enrolment, at enrolment, and then at weeks 4, 12, 24, 40, and 52 (and week 72 and 104 in Uganda); buffy coat was obtained at enrolment, and at weeks 24, 40, and 52 (and week 104 in Uganda). The HIV-1 testing algorithm comprised parallel HIV-1 rapid tests at the clinics, with discordant or positive tests after enrolment triggering ELISA testing at local laboratories and confirmation at a central laboratory in South Africa. A second serum sample was obtained at the subsequent visit after a first positive rapid test result. The central laboratory analysed samples from the visit at which a positive rapid test result was obtained and from all previous visits at which samples were obtained. This analysis allowed detection of seroconversion to be established as at or before enrolment.
The algorithm was designed to confirm HIV-1 infection on the basis of two separate samples, with two different methods of diagnosis. Serum samples were tested for HIV-1 antibodies with Abbott AxSYM HIV Ag/Ab Combo (fourth-generation ELISA; Wiesbaden, Germany), Bio-Rad Genetic Systems HIV-1 ELISA (third-generation ELISA; Redmond, WA, USA), and Genetic Systems HIV-1 Western Blot (Redmond, WA, USA) assays. We used Biomerieux Vironostika HIV-1 antigen ELISA (Boxtel, Netherlands) as a confirmatory assay for p24 testing. Buffy coat samples were tested with the Roche qualitative DNA PCR Version 1.5 assay (Roche Diagnostic Systems, Branchburg, NJ, USA). The Roche COBAS Amplicor HIV-1 Monitor (Roche Diagnostic Systems) was used for the detection of HIV-1 RNA if the buffy coat specimen was not satisfactory.
The primary efficacy endpoint was HIV-1 infection, confirmed by the central reference laboratory, in participants who were confirmed without HIV-1 infection at enrolment. Secondary efficacy endpoints were acquisition of herpes simplex virus type 2 (HSV-2) infection by participants who were HSV-2 seronegative at enrolment, which was established serologically and confirmed by the central laboratory at 40 weeks and 52 weeks; or presence of Neisseria gonorrhoeae or Chlamydia trachomatis, which was established by a positive nucleic acid amplification assay at 24 weeks.
The primary safety endpoint was a grade 3 (severe) or worse clinical or laboratory adverse event, irrespective of relation to trial gel. Secondary safety endpoints were reported local toxic effects (any grade of genital itching, burning, internal epithelial disruption, internal erythema, or internal oedema) and systemic toxic effects (any increase in grade from baseline in routine laboratory variables). We systematically assessed these outcomes because of a probable or known association with PRO2000 when administered vaginally or systemically in previous trials.21,22
The primary efficacy outcome was originally designed to be measured at 40 weeks, which changed to 52 weeks after commencement of the trial.18
The MDP301 trial protocol stated that the Independent Data Monitoring Committee could recommend early termination of the trial “if there was proof beyond reasonable doubt that one of the trial interventions is clearly indicated or clearly contraindicated in terms of a net difference in seroincidence or adverse events”. The 2% PRO2000 gel was discontinued on Feb 14, 2008, after a review by the committee on Feb 8, 2008 of available data up to Jan 15, 2008. The committee advised there was little chance of 2% PRO2000 gel showing benefit given the planned sample size and postulated effect size. However, the conditional power for significant benefit from the 0·5% PRO2000 dose, based on the original sample size assumptions, was sufficiently high to warrant trial continuation. We report data for the 0·5% PRO2000 and placebo groups to study end, and data for the 0·5% PRO2000, 2% PRO2000, and placebo groups with data up until discontinuation on Feb 14, 2008.
MDP301 was designed with 80% power to detect a 35% reduction in HIV-1 incidence (90% for a 40% reduction), assuming an incidence in the placebo group of 4·0 per 100 woman-years. The assumptions made for incidence and loss to follow-up (we predicted 3300 participants would be needed per group, assuming 20% loss of woman-years of follow-up) were derived from data in previous cohort studies.18
The primary efficacy analysis comprised all enrolled participants, excluding those with HIV-1 infection at enrolment, those without follow-up data for HIV infection, and with censoring at 52 weeks (plus 6 week window for final visit) and while gel use was discontinued because of pregnancy. For women who did not have HIV-1 infection when gel use was resumed after pregnancy, additional time was added to the woman-years of follow-up for the primary efficacy analysis.
A second efficacy analysis was done with the same criteria as the primary efficacy analysis, but without censoring for pregnancy and using all follow-up data. We assessed secondary efficacy endpoints with two further analyses, censoring at 24 and 40 weeks (plus 4-week window) from enrolment, excluding those with HIV-1 infection at enrolment and censoring for pregnancy.
We did two planned subgroup analyses of patients, including tests for interaction. One subgroup analysis was stratified by research centre and one was done with postrandomisation data and categorised women according to the consistency of gel use, with the expectation that efficacy against HIV-1 transmission would be greater in consistent users than it was in sporadic users. Gel use was predefined as consistent if women reported use during the last sex act at 12 (92%) or more of 13 visits, or at least 92% of visits attended if fewer than 13; returned at least one used applicator to support their answer when appropriate; and attended at least seven of the expected 13 visits (unless they became pregnant or were infected with HIV-1 during follow-up).
In patients with HIV-1 seroconversion, the date of detection of HIV-1 infection was established by an endpoint committee. Woman-years of observation were censored at the date of seroconversion, estimated by the midpoint between the last negative test and date of detection, or at the last HIV-1-negative test for patients who did not become infected.
We analysed the primary efficacy endpoint as time-to-event, and groups were compared by use of hazard ratios (HRs) relative to the placebo group, 95% CI, and p values, which were obtained by Cox proportional hazards regression and stratified by clinic. We analysed secondary efficacy endpoints as binary outcomes by logistic regression, the proportion infected with HSV-2 at week 40 and week 52 for those HSV-2 negative at baseline; and cross-sectional prevalence of N gonorrhoeae and C trachomatis for all patients tested at week 24.
All women with follow-up clinical data were included in the safety analyses. Safety endpoints were analysed as time-to-first-event and groups compared as for the efficacy analysis. All analyses for the 2% PRO2000 group were undertaken with all three groups, and were censored when the 2% PRO2000 gel was discontinued on Feb 14, 2008. All statistical tests were two-sided and all analyses were done with Stata version 10.1.
Role of the funding source
MDP301 was supported by the UK Department for International Development (DFID), the UK Medical Research Council (MRC), the European and Developing Countries Clinical Trials Partnership, and the International Partnership for Microbicides. The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Endo Pharmaceuticals Solutions donated the study gels, provided regulatory support, and participated in design and management of the study. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.