Mean follow up was 20.4 months and overall study retention, defined as the proportion of those enrolled (except the window period infections) who had a study exit visit with an HIV test, was 93.6%. Of the 5258 person-years of follow-up accumulated on 3050 women (Fig. 1), 240 person-years (6.1%) in 620 women comprised follow-up during which study product was temporarily withheld, mostly due to pregnancy, in accordance with the study protocol. Women reported using gel in 81.1% of last sex acts. Gel adherence was similar in the three gel arms. Self-reported condom use during the last sex act was similar in the three gel arms, but higher in the no gel arm (71.7 vs. 80.7%, P = <0.0001). Overall, women reported gel use in conjunction with condoms during study follow-up in 61.3% of sex acts. Gel was used in 69.1% of last acts in which a condom was not used.
The HIV incidence rate per 100 person-years was 4.1 (54 of 1304) in the BufferGel arm, 2.7 (36 of 1332) in the 0.5% PRO2000 gel arm, 3.9 (51 of 1305) in the placebo gel arm, and 4.0 (53 of 1318) in the no gel arm (Table 2). The hazard ratio of HIV incidence rates in 0.5% PRO2000 gel arm compared with the placebo gel arm was 0.70 (P = 0.10) and with the no gel arm was 0.67 (P = 0.06). Figure 2 shows how the Kaplan–Meier survival curves from HIV infection in the 0.5% PRO2000 gel arm differs from the remaining three study arms. The HIV incidence rate in the 0.5% PRO2000 gel arm was lower, though not statistically significant, than the rate in the placebo gel arm at seven of the eight sites (Table 3). In the per-protocol analysis that excludes follow-up beyond 2 months after initiating product hold, the hazard ratio of HIV incidence rates in the 0.5% PRO2000 gel arm was 0.71 (P = 0.13) compared with the placebo gel arm and 0.64 (P = 0.04) compared with the no gel arm. There was no difference in HIV incidence between the BufferGel and both control arms (Table 3).
The hazard ratio of the HIV incidence rates in the placebo gel arm compared with the no gel arm was 0.97 (P = 0.89), reflecting the similarities in the HIV incidence rates in the placebo gel arm [3.9 per 100 person-years, 95% confidence interval (CI) 2.9–5.1] and in the no gel arm (4.0 per 100 person-years, 95% CI 3.0–5.3). There was little change in this hazard ratio after adjusting for baseline sexual behavior and participant characteristics.
In order to assess whether self-reported PRO2000 gel use was associated with a lower rate of HIV, a subgroup analysis stratified low and high gel users at the median; women with 85% or more gel use in their quarterly reported last sex acts were categorized as high gel users, whereas those with less than 85% gel use were categorized as low gel users. The HIV incidence rate among low gel users was 3.0 per 100 person-years (18 of 592) in the 0.5% PRO2000 gel group and 3.3 per 100 person-years (19 of 568) in the placebo gel group (hazard ratio = 1.04, 95% CI 0.55–2.00). However, the HIV incidence rate among high gel users was 2.4 per 100 person-years (18 of 740) in the 0.5% PRO2000 gel group and 4.3 per 100 person-years (32 of 738) in the placebo gel group (hazard ratio = 0.55, 95% CI 0.31–0.98). In the low condom use (less than 85% condom use in quarterly reported last sex acts) subgroup of the high gel users, the HIV incidence rate was 1.0 per 100 person-years (three of 299) in the 0.5% PRO2000 gel group and 4.6 per 100 person-years (15 of 324) in the placebo gel group (hazard ratio = 0.21, 95% CI 0.06–0.73).
After adjusting for multiple comparisons, there were no statistically significant differences in systemic and local adverse events among the four study arms in the intent-to-treat and per-protocol analyses. Overall, the incidence rate of deep epithelial disruption was 1.55 per 100 person-years, with rates being similar across the four study arms (Table 4). The higher incidence of blood in the vagina with no identified source (Table 4) in the 0.5% PRO2000 gel arm was statistically significant compared with the placebo gel arm (3.5 vs. 1.8, P < 0.01), but not compared with the no gel arm (3.5 vs. 2.3, P = NS). After adjusting for multiple comparisons, these differences were no longer statistically significant. A total of 613 pregnancies occurred during follow-up in the study yielding an overall pregnancy rate of 11.3 per 100 person-years. Pregnancy rates were similar across the four study arms. The lowest pregnancy rate was 9.2 per 100 person-years (95% CI 7.0–11.4) at the combined Harare and Chitungwiza sites in Zimbabwe and the highest was 16.5 per 100 person-years (95% CI 13.4–19.5) at the Blantyre site (Table 4).
The overall incidence rates of infection with Neisseria gonorrheae, Chlamydia trachomatis, Trichomonas vaginalis, and Treponema pallidum, as well as bacterial vaginosis were 1.6, 3.9, 15.3, 0.9, and 160.5 per 100 person-years, respectively. These infection rates were similar in the four study arms.
The HPTN 035 trial showed that 0.5% PRO2000 gel was well tolerated and, compared with the placebo gel, reduced the incidence of HIV infection by a modest 30%, although this finding was not statistically significant (P = 0.10). The consistent presence of the 0.5% PRO2000 effect on HIV infection against each of the two comparator arms and in almost all study sites, the greater protection observed in self-reported high PRO2000 gel users compared with low gel users, and the biological plausibility from animal challenge studies [24,25] suggested a potentially promising signal on PRO2000. However, these data alone were insufficient to conclude that 0.5% PRO2000 gel protected against HIV infection. Subsequently, our trial's initial encouraging signal was superseded by the finding of no protection against HIV infection in the phase III MDP301 trial .
The HEC placebo gel  was found to be well tolerated and had no demonstrable effect on HIV infection when compared with the no gel arm, even after adjusting for baseline characteristics including condom use. This addressed a key concern in microbicide research and makes HEC a suitable ‘universal’ placebo for future microbicide gel trials .
The results of microbicide effectiveness trials are impacted by several inherent design and implementation challenges , including lengthy periods off-product (mainly due to pregnancy), adherence, other sexually transmitted infections, unprotected and unreported anal sex, use of intravaginal substances that may interfere with the study gel, and difficulties in applying study gel as prescribed.
The 6.1% of follow-up time that was off-product, mainly due to the 11.3% pregnancy rate, had little, if any, effect in the placebo gel comparisons. However, it had a small but important impact in the comparisons with the no gel arm in which the effect of PRO2000 changed from 33% (P = 0.06) in the intent-to-treat analysis to 36% (P = 0.04) in the per-protocol analysis, highlighting the potential impact of even relatively modest pregnancy rates on microbicide trial outcomes. Adherence is a major challenge in microbicide trials . In the recent Carraguard trial, an applicator dye test revealed a much lower estimate of use compared with self-reported adherence (42.1 vs. 96.1%) . Our trial did not have such an objective measure of adherence because the dye test performed poorly on the applicator used in this trial . To increase their chances of success, future microbicide trials will need to enroll a higher proportion of women who will maintain high adherence for both study gel and reliable contraception during the trial.
Although interpretation of microbicide trial results can be complicated by the indirect effect of other sexually transmitted infections on HIV infection, this did not apply to this trial, as the three study gels did not alter the risk of other sexually transmitted infections. Self-reported unprotected anal sex, intravaginal substance use, and concerns about the 1-h presex insertion requirement were low and, therefore, unlikely to have had an impact on the trial result. However, these may have been under-reported, making it difficult to estimate their full potential impact on the study outcome.
BufferGel was found to be well tolerated, but did not alter the risk of HIV infection. This could be due to the differential effect of acidity and BufferGel on cell-free  compared with cell-associated viruses . Moreover, the duration of action of BufferGel is brief . It was posited that BufferGel would reduce HIV susceptibility by reducing the prevalence of bacterial vaginosis, as observed in a phase I trial ; however, no effect on bacterial vaginosis was observed in this trial, possibly due to lower gel use here compared with twice-daily use in the phase I trial.
The pregnancy rates in all arms were similar. Although HPTN 035 was not specifically designed to assess contraceptive efficacy due to the high background rates of effective contraception use in the study population, 0.5% PRO2000 gel and BufferGel did not demonstrate a contraceptive effect in this study.
Viewed jointly, the HPTN 035 and MDP 301 trials suggest that 0.5% PRO2000 gel may have little or no effect on reducing a woman's risk of HIV infection. Hope is now being placed in the topical use of antiretroviral agents, like tenofovir gel , as the next class of candidate microbicides and on new formulations to improve adherence, like vaginal rings. Most recently, results from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 trial showed that tenofovir gel had a protective effect of 39% against HIV . The protective efficacy of tenofovir gel has demonstrated that microbicides can prevent HIV infection and could potentially alter the course of the HIV epidemic . Although efforts to bring tenofovir gel into widespread public health use are underway, there are limitations to using prescription-only medications, including their potential for drug resistance, and potential adverse effects on concomitant viral infections such as hepatitis B. For these reasons, the microbicide field should not abandon the search for a well tolerated, single use with sex, over-the-counter microbicide, as had been hoped for when we undertook this study of PRO2000 gel and BufferGel.
HIV Prevention Trials Network (HPTN) 035 was funded by the US National Institutes of Health (NIH). The study was designed and implemented by the HPTN and the Microbicide Trials Network (MTN). The HPTN (U01AI46749) has been funded by the National Institute of Allergy and Infectious Diseases (NIAID), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Drug Abuse (NIDA), and National Institute of Mental Health (NIMH). The MTN (U01AI068633) has been funded by NIAID, NICHD, and NIMH. The study products were provided free of charge by Endo Pharmaceuticals and ReProtect Inc. The US Agency for International Development (USAID) provided funding for manufacturing of BufferGel for this study. The Statistical Center was supported by NIAID (UO1AI068615). We specially acknowledge Dr Roberta Black for her unstinting support and encouragement from conception to completion of this trial, without which this study would not have been possible.
The HPTN 035 study team includes the following given below:
Sponsors: US National Institutes of Health (R.B., L.S.-T., and S.E.).
Endo Pharmaceuticals Solutions Inc. (A.P.).
Protocol Chair: S.S.A.K.
Blantyre, Malawi: T.T., N.K., B.M., S.H., C.N., E.K.
Durban and Hlabisa, South Africa: G.R., R.G., N.C., N.D.-Q., S.G., N.M.
Harare, Zimbabwe: Z.M.C., N.P., A.v.d.S., T.M., M.M., N.M.
Lilongwe, Malawi: I.F.H., F.M., T.T., L.C., T.M.
Lusaka, Zambia: G.P., M.K., C.R., M.K., A.B.
Philadelphia, USA: L.M., J.P., N.T., S.W., E.Y.
Coordinating Center: W.C., A.C., K.G., R.W.
Statistical Center: M.C., C.K., C.M., B.M., B.A.R., T.F.
Network Laboratory: S.L.H., E.P.-M., L. Rabe.
The study was conceptualized and designed by S.S.A.K., B.A.R., G.R., I.F.H., Z.M.C., T.T., L.M., A.C., A.P., T.R.M., B.M., S.L.H., L.S.-T. Data was gathered by I.F.H., Z.M.C., T.T., M.K., L.M., B.M. Data was analyzed and interpreted by S.S.A.K., I.F.H., Z.M.C., T.T., M.K., L.M., A.C., E.P.-M., B.M. S.S.A.K. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. S.S.A.K. took responsibility for writing the article and all co-authors contributed to critical revision of the article.
T.R.M. is an employee of ReProtect that sponsored BufferGel for the study; and A.P. was an employee of, and held an equity interest in, Endo Pharmaceuticals Solutions (formerly Indevus Pharmaceuticals), the owner of PRO2000 gel. L.S.-T. is an employee of the National Institutes for Health, which funded this study. None of the other authors have any specified relationships with any companies that might have an interest in the submitted work in the previous 3 years; their spouses, partners, or children have no financial relationships that may be relevant to the submitted work; and S.S.A.K., B.A.R., G.R., I.F.H., Z.M.C., T.T., M.K., L.M., A.C., E.P.-M., B.M., S.L.H. have no nonfinancial interests that may be relevant to the submitted work.
2. Davis KR, Weller SC. The effectiveness of condoms in reducing heterosexual transmission of HIV. Fam Plan Perspect 1999; 31:272–279.
3. Elias CJ, Coggins C. Female controlled methods to prevent sexual transmission of HIV. AIDS 1996; 10:S43–S51.
4. Elias CJ, Heise LL. Challenges for the development of female-controlled vaginal microbicides [editorial]. AIDS 1994; 8:1–9.
5. Stone AB, Hitchcock PJ. Vaginal microbicides for preventing the sexual transmission of HIV. AIDS 1994; 8:S285–S293.
6. Kreiss J, Ngugi E, Holmes K, Ndinya-Achola J, Waiyaki P, Roberts PL, et al
. Efficacy of nonoxynol 9 contraceptive sponge use in preventing heterosexual acquisition of HIV in Nairobi prostitutes. JAMA 1992; 268:477–482.
7. Roddy RE, Zekeng L, Ryan KA, Tamoufe U, Weir SS, Wong EL. A controlled trial of nonoxynol 9 film to reduce male-to-female transmission of sexually transmitted diseases. N Engl J Med 1998; 339:504–510.
8. Richardson BA, Lavreys L, Martin HLJ, Stevens CE, Ngugi E, Mandaliya K, et al
. Evaluation of a low-dose nonoxynol-9 gel for the prevention of sexually transmitted diseases: a randomized clinical trial. Sex Transm Dis 2001; 28:394–400.
9. Roddy RE, Zekeng L, Ryan KA, Tamoufe U, Tweedy KG. Effect of nonoxynol-9 gel on urogenital gonorrhea and chlamydial infection: a randomized controlled trial. JAMA 2002; 287:1117–1122.
10. Van Damme L, Ramjee G, Alary M, Vuylsteke B, Chandeying V, Rees H, et al
. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 2002; 360:971–977.
11. Feldblum PJ, Adeiga A, Bakare R, Wevill S, Lendvay A, Obadaki F, et al
. SAVVY vaginal gel (C31G) for prevention of HIV infection: a randomized controlled trial in Nigeria. PLoS One 2008; 3:e1474.
12. Van Damme L, Govinden R, Mirembe FM, Guedou F, Solomon S, Becker ML, et al
. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 2008; 359:463–472.
13. Skoler-Karpoff S, Ramjee G, Ahmed K, Altini L, Plagianos MG, Friedland B, et al
. Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial. Lancet 2008; 372:1977–1987.
14. Mayer KH, Abdool Karim SS, Kelly C, Maslankowski L, Rees H, Profy AT, et al
. Safety and tolerability of vaginal PRO 2000 gel in sexually active HIV-uninfected and abstinent HIV-infected women. AIDS 2003; 17:321–329.
15. Mayer KH, Peipert J, Fleming T, Fullem A, Moench T, Cu-Uvin S, et al
. Safety and tolerability of BufferGel, a novel vaginal microbicide, in women in the United States. Clin Infect Dis 2001; 32:476–482.
16. Van Damme L, Wright A, Depraetere K, Rosenstein I, Vandersmissen V, Poulter L, et al
. A phase I study of a novel potential intravaginal microbicide, PRO 2000, in healthy sexually inactive women. Sex Transm Infect 2000; 76:126–130.
17. Smita J, Soma D, Beverly B, Albert P, JoAnn K, Fang G, et al
. Phase I safety study of 0.5% PRO 2000 vaginal Gel among HIV un-infected women in Pune, India. AIDS Res Ther 2006; 3:4.
18. Bentley ME, Morrow KM, Fullem A, Chesney MA, Horton SD, Rosenberg Z, et al
. Acceptability of a novel vaginal microbicide during a safety trial among low-risk women. Fam Plan Perspect 2000; 32:184–188.
19. van De Wijgert J, Fullem A, Kelly C, Mehendale S, Rugpao S, Kumwenda N, et al
. Phase 1 trial of the topical microbicide BufferGel: safety results from four international sites. J Acquir Immune Defic Syndr 2001; 26:21–27.
20. Olmsted SS, Dubin NH, Cone RA, Moench TR. The rate at which human sperm are immobilized and killed by mild acidity. Fertil Steril 2000; 73:687–693.
21. McCormack S, Ramjee G, Kamali A, Rees H, Crook AM, Gafos M, et al
. PRO2000 vaginal gel for prevention of HIV-1 infection (Microbicides Development Programme 301): a phase 3, randomised, double-blind, parallel-group trial. Lancet 2010; 376:1329–1337, doi: 10.1016/S0140-6736(10)61086-0.
23. Fleming TR, Richardson BA. Some design issues in trials of microbicides for the prevention of HIV infection. J Infect Dis 2004; 190:666–674.
24. Lewis MG, Wagner W, Yalley-Ogunro J, Greenhouse J, Profy AT. Efficacy of PRO 2000 Gel in a macaque model for vaginal HIV transmission [abstract A-191]
. In: Microbicides
2002; 12–15 May 2002; Antwerp, Belgium.
25. Weber J, Nunn A, O'Connor T, Jeffries D, Kitchen V, McCormack S, et al
. ‘Chemical condoms’ for the prevention of HIV infection: evaluation of novel agents against SHIV(89.6PD) in vitro and in vivo. AIDS 2001; 15:1563–1568.
26. Tien D, Schnaare RL, Kang F, Cohl G, McCormick TJ, Moench TR, et al
. In vitro and in vivo characterization of a potential universal placebo designed for use in vaginal microbicide clinical trials. AIDS Res Hum Retroviruses 2005; 21:845–853.
27. Lagakos SW, Gable AR, editors. Methodological challenges in biomedical HIV prevention trials
. Washington, District of Columbia: National Academy of Sciences; 2008.
28. Abdool Karim SS, Baxter C. PRO 2000: next steps for microbicides. Future Virol 2009; 4:317–320.
29. Austin MN, Rabe LK, Hillier SL. Limitations of the dye-based method for determining vaginal applicator use in microbicide trials. Sex Transm Dis 2009; 36:368–371.
30. Connor RI. Sensitivity of nonclade B primary HIV-1 isolates to mildly acidic pH. J Acquir Immune Defic Syndr 2006; 43:499–501.
31. Olmsted SS, Khanna KV, Ng EM, Whitten ST, Johnson ON 3rd, Markham RB, et al
. Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model. BMC Infect Dis 2005; 5:79.
32. Zeitlin L, Hoen TE, Achilles SL, Hegarty TA, Jerse AE, Kreider JW, et al
. Tests of Buffergel for contraception and prevention of sexually transmitted diseases in animal models. Sex Transm Dis 2001; 28:417–423.
33. Abdool Karim Q, Abdool Karim SS, Frohlich JA, Grobler AC, Baxter C, Mansoor LE, et al
. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010; 329:1168–1174.
34. Williams BG, Abdool Karim SS, Gouws E, Abdool Karim Q. Potential impact of tenofovir gel on the HIV epidemic in South Africa
. XVIII International AIDS Conference
. Vienna, Austria, 2010.
Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
BufferGel; HIV prevention; microbicide; PRO2000 gel; women