Some Candidate Microbicides Can Damage Epithelia
The candidate microbicide cellulose sulfate (CS) can disrupt epithelial integrity and make it easier for HIV particles to cross epithelial barriers, according to a study by researchers at Albert Einstein College of Medicine in New York City. The results, still unpublished but presented earlier this year at the Conference on Retroviruses and Opportunistic Infections in Boston and at the Microbicides 2008 conference in New Delhi, offer a possible explanation for the association between use of CS and increased HIV acquisition in a recent clinical trial. Two trials testing the ability of CS gels to prevent HIV transmission were halted last year because one trial showed a trend toward higher HIV infections in some women who received CS, as compared to placebo.
The study, led by Betsy Herold of Albert Einstein College of Medicine, found that CS can damage epithelial layers. Herold and colleagues also analyzed three other compounds. The spermicide Nonoxynol-9 (N-9) showed even greater epithelial disruption than CS, consistent with clinical trial results that found N-9 also tends to increase susceptibility to HIV infection in women (Lancet 360, 971, 2002). Two other microbicide candidates, PRO 2000 and the antiretroviral tenofovir, did not show the same disruptive effects when applied in the same concentrations.
The researchers measured the effects of these compounds on an in vitro cultured epithelial cell layer—made of either uterine epithelial cells or of reconstituted vaginal tissue—by measuring the transepithelial resistance to an electric current. As long as the integrity of these epithelia remains intact, transepithelial resistance should remain high. The researchers found that an 18-hour exposure to either N-9 or CS led to a drop in resistance, while PRO 2000 and tenofovir had little effect. CS is a polymer composed of sugar molecules with sulfate groups. It is unclear why PRO 2000, which is structurally similar to CS, does not have the same detrimental effects on epithelia. However, Herold says, preliminary data from her lab suggest that CS and PRO 2000 might activate different signaling pathways. Use of N-9 and CS also made the cultured epithelium leaky, allowing HIV particles to more easily cross the epithelium.
Confocal microscopy of the cultured reconstituted vaginal tissue showed that CS treatment led to a marked loss of syndecan and desmoglein, proteins that are important for epithelial integrity. There was also downregulation of the RNA expression of the genes encoding such proteins.
CS also increased the expression of interleukin (IL)-6 in the cultured epithelial cells and induced a three-fold increase in activation of the transcription factor NF-κB in peripheral blood mononuclear cells (PBMCs), suggesting that it caused an inflammatory response.
The researchers also administered the microbicide gels to mice daily for one week and then analyzed their genital tracts. Both N-9 and CS caused mice to be more susceptible to HSV infection.
Preclinical and Phase I assessments of CS did not uncover its detrimental effects on epithelia. Currently, the FDA recommends microbicide candidates be tested preclinically in rabbits, Herold says. In this model, 10 daily doses of a candidate microbicide are applied vaginally followed by histological analysis of the vaginal tissues. Phase I trials assess subjective symptoms, such as whether a microbicide burns, and use colposcopy, which looks at visible disruptions of the cervix, Herold says. These assessments, however, missed the effects observed in this study.
“We propose that epithelial integrity, inflammatory response, and susceptibility to infection are included in preclinical testing of candidate microbicides,” concludes Pedro Mesquita, who was also involved in the study.
The other two microbicide candidates evaluated in this study, PRO 2000 and tenofovir, are currently in clinical trials. A Phase III trial in women in South Africa, Tanzania, Uganda, and Zambia is testing safety and effectiveness of PRO 2000 in preventing HIV infection in women and is sponsored by the UK’s Medical Research Council. That trial’s independent data monitoring committee recommended discontinuing the high-dose arm (testing a 2% dose of PRO 2000) earlier this year due to lack of efficacy, but the low-dose arm (using an 0.5% dose of PRO 2000) is ongoing. The National Institute of Allergy and Infectious Diseases is also testing safety and effectiveness of the low dose of PRO 2000 in a Phase II/IIb trial called HPTN 035 in Malawi, South Africa, Zambia, Zimbabwe, and the US. A microbicide gel formulation of tenofovir is also currently being tested in a Phase II trial (HPTN 059).
IOM Report Addresses the Challenges of HIV Prevention Trials
The prestigious US Institute of Medicine (IOM), an independent advisory group on public health policy, convened a series of meetings last year on the methodological challenges of conducting non-vaccine HIV prevention trials (see Advisory Panel considers complexities of HIV prevention trials, IAVI Report, January-February 2007 andOptimizing HIV prevention research, IAVI Report, March-April 2007). The final report based on these proceedings, as well as site visits by IOM committee members to clinical trial sites in Uganda and South Africa, was issued in February (www.nap.edu/catalog/12056.html). The report outlines the recent spate of late-stage clinical trials in the HIV prevention field that have failed to provide any benefit in reducing the risk of HIV infection, leading the authors to conclude that, “A near-perfect biomedical intervention for preventing HIV infection is unlikely to be available in the near future.”
The IOM committee was organized at the request of the Bill & Melinda Gates Foundation and was to provide recommendations on how future trials of microbicides and pre-exposure prophylaxis (see Treatment as Prevention, IAVI Report, May-June 2006) could be conducted in a way that could increase the likelihood of success and enable donors to optimally invest their limited financial resources. Stephen Lagakos, director of the Center for Biostatistics at Harvard University, chaired this committee and co-authored the report with Alicia Gable of the IOM. The two also recently wrote a perspective article in the New England Journal of Medicine summarizing the report’s main conclusions (N. Engl. J. Med. 358, 1543, 2008). “Shortcomings in research design have inhibited progress in identifying effective HIV-prevention interventions,” the authors said.
One shortcoming the report focused on in particular was the way HIV incidence has been estimated in advance of some efficacy trials (see Moving Targets, IAVI Report, May-June 2007). The IOM committee recommended that all late-stage trials be designed based on incidence estimates collected through traditional cohort follow-up studies in the communities where the trial will occur, and that these estimates should be corroborated by at least one other source.
High pregnancy rates during HIV prevention trials, and the impact on retention of female volunteers, was another critical issue addressed in the report. Female volunteers are typically not allowed to receive the experimental intervention—microbicide gel or antiretroviral drug—during pregnancy because of potential safety risks to the fetus, but their exclusion can confound results. On this issue, the authors suggested researchers should try to determine the safety of the intervention in pregnant women as best as possible before initiating the trial to determine circumstances in which women could potentially continue to participate in HIV prevention trials even during pregnancy.
The report also outlines several other ways that trials can be designed to determine the influence individual behavior and adherence have on the final results, which is critical in non-vaccine HIV prevention trials.
Microbicide Trials Update
In February the Population Council announced that the microbicide gel Carraguard, which was tested in a randomized, double-blind, placebo-controlled Phase III trial, had no effect on HIV infection rates in women. The trial was conducted at three sites in South Africa and involved 6,202 women between the ages of 16 and 72. Final results showed that 134 women who received Carraguard became HIV infected, compared to 151 placebo recipients.
Carraguard contains the compound carrageenan, a seaweed derivative commonly used as a stabilizer and thickening agent in food and cosmetics. Prior to initiating the Phase III trial, the Population Council conducted two Phase II safety studies of Carraguard in South Africa and Thailand, involving a total of 565 HIV-uninfected women.
A critical aspect of the Carraguard trial was adherence. Women were counseled to apply the microbicide before every sex act, and although the self-reported adherence rates were 96%, researchers estimate that the actual adherence was much lower. “It’s possible that low levels of adherence in the trial were responsible for why the product didn’t show an effect,” says Barbara Friedland of the Population Council.
To measure adherence, researchers collected behavioral information directly from participants and also treated the microbicide applicator with a compound that, after analysis, indicated whether or not the applicator had been in contact with vaginal mucous. The results of these tests showed women used the gel in only 44% of sex acts, and only 10% were estimated to have used it during every sex act.
An applicator test is one method researchers are using to better estimate adherence, but even this approach is complicated. “All we can tell is whether the applicator was inserted in the vagina or not,” Friedland says. “We don’t know when in relation to the sex act the applicator was inserted.”
Other updates on microbicide trials occurred during the Microbicide 2008 Conference, which was held in New Delhi from February 24-27. Researchers presented data from a Phase II safety study, known as HPTN 059, which tested a gel formulation of tenofovir, an antiretroviral (ARV) commonly used in the treatment of HIV infection. The findings, presented by Sharon Hillier of the Microbicide Trials Network, revealed that the product was safe for daily use in sexually-active, HIV-uninfected women. This trial evaluated both daily use of the microbicide and a sex-dependent regimen, in which women were instructed to only apply the microbicide just prior to sex. Adherence was based on self-reported behavior and researchers estimated that 80% of the women in the sex-dependent group adhered to the regimen, compared to 83% of the women in the daily-use group. This candidate is one of several ARV-based microbicides that are currently being tested.
Vaccine Briefs written by Andreas von Bubnoff, Kristen Jill Kresge, and Alix Morris, contributing writer