To the Editors:
There is a need to improve in vitro testing to evaluate topical microbicide candidates that prevent acquisition of HIV. Many vaginal microbicides with different anti-HIV activities have undergone preclinical testing and a few of those have been selected for clinical safety and efficacy testing. Clinical efficacy trials of vaginal microbicide gels have yielded mixed results. Initial nonspecific entry inhibitors were shown to be ineffective in clinical efficacy trials,1–4 whereas more recent testing of microbicide gels containing the nucleoside reverse transcriptase inhibitor, tenofovir, has shown some level of protection in 1 of 2 clinical trials.5,6 Yet, nearly all preclinical testing outcomes predicted that products should significantly reduce sexual acquisition of HIV when used appropriately in a clinical trial. However, a recent report using ex vivo testing of the microbicide Pro2000 demonstrated that this product loses anti-HIV activity after vaginal application and sexual intercourse.7 Changes in anti-HIV activity over time after vaginal application have not been studied for most candidate vaginal microbicides.
We investigated the carrageenan-based vaginal gel Carraguard, an HIV entry inhibitor that failed in a clinical efficacy trial,1 for degradation and loss of anti-infective activity after vaginal application. Cervicovaginal lavages (CVLs) were collected from 16 HIV-infected Thai women participating in a randomized, controlled, 3-treatment (Carraguard, methylcellulose placebo, no product) crossover safety trial.8 Women applied each treatment daily for 7 days after menses. The order of the 3 treatments was randomized. CVLs (5 mL) were collected on the first clinic visit in each treatment cycle before gel application (T0), 15 minutes after gel application (T15min), and on day 7 clinic visit which was 8–24 hours after the final gel application (T8–24hr). Self-reported adherence was 98% overall, and participants reported that vaginal application of the gel was highly acceptable.9
Carrageenan, the active ingredient of Carraguard, was measured in CVLs using a modified methylene blue assay.10 Each CVL was diluted 1:26 in distilled water, mixed 10:1 with a 0.4-mM methylene blue aqueous solution, and its OD540nm was compared with those in a standard curve of carrageenan dilutions (lower limit of detection, 25 μg/mL). Carrageenan was detected in CVLs from all 16 women at T15min and from 12 women at T8–24hr. Carrageenan levels in CVLs were significantly higher at T15min (median: 304 μg/mL; range, 103–3154 μg/mL) compared with T8–24hr (median: 98 μg/mL, range, <25–282 μg/mL) (Mann–Whitney, P < 0.001). Carrageenan was not detected in CVLs at T0 nor in the CVLs collected after vaginal application of methylcellulose placebo or no product.
CVLs were then tested for carrageenan anti-infective activity using an ex vivo titer reduction assay. HIV-1 susceptible TZM-bl cells were plated in microtiter wells, briefly incubated in CVL supernatant, then virus-challenged with serial dilutions of a CCR5-tropic CRF01_AE HIV-111 using a standard infectious titer protocol12 that reports 50% tissue culture infectious dose (TCID50) values. The virus titer reduction value was the difference between TCID50 from CVL samples at follow-up (T15min and T8–24hr) and baseline (T0). It was calculated for each woman and by type of treatment. The T0 CVLs produced a narrow range of baseline TCID50 values (4.67–5.55 log10, median 4.85 log10) that were slightly higher than those produced using normal cell culture media (median 4.67 log10, P < 0.001). All 16 T15min CVLs collected after Carraguard application produced a reduction in TCID50 from 1 to >4.5 log10 and 12 of 16 CVLs at T8–24hr after Carraguard use reduced TCID50 from 1 to 2.5 log10 (Fig. 1). Importantly, 3 of 4 CVLs with carrageenan levels more than 600 μg/mL at T15min completely prevented target cell HIV infection, and all 4 CVLs at T8–24hr with undetectable carrageenan levels showed no anti-infective activity. There was a strong correlation between a carrageenan level of CVL and its anti-infective activity (Spearman, P < 0.001, data not shown). In T15min and T8–24hr CVLs with similar carrageenan levels (100–300 μg/mL), there was no significant difference between their anti-infective activities in the titer reduction assay. CVLs collected after methylcellulose use (Fig. 1) had no measurable anti-infective activity. Native infectious virus was not detected in CVLs of the HIV-infected women, including those collected when women were using methylcellulose placebo or no product (data not shown).
Our ex vivo evaluation of anti-HIV activity indicates that Carraguard microbicide gel retained some ability to reduce HIV infection of target cells from 15 minutes up to 8–24 hours after vaginal application. Although carrageenan levels decreased in CVLs at T8–24hr, their level of anti-infective activity ex vivo was not significantly different from CVLs at T15min with similar concentrations of carrageenan. Thus, the difference over time in anti-infective activity measured ex vivo was likely because of the reduction in recoverable carrageenan and not product inactivation in vivo. A similar result was reported for Pro2000 after vaginal application and sexual intercourse.7
The failure of Carraguard to prevent sexual acquisition of HIV-1 in a clinical efficacy trial was likely because of multiple factors including low adherence (41% of sex acts).1 Nonetheless, data from our ex vivo assay suggests that carrageenan was present for at least 8–24 hours after vaginal application and retained some anti-infective activity. However, the reduction in genital tract carrageenan levels over time suggests that decreasing amounts of product at the cervical–vaginal mucosa might not have been sufficient to provide durable protection from virus in the semen of an infected male sexual partner. The carrageenan levels we measured produced only an infectious titer reduction, not complete protection from infection, in 13 of 16 CVLs at T15min and in all 16 CVLs at T8–24hr. In addition, the effectiveness of carrageenan-based microbicide gels in our ex vivo assay would likely be reduced further in the presence of semen as has previously been reported for other polyanion-based HIV-entry inhibitors.13 Limitations of this study are the lack of controls for possible variations in how CVLs were collected and that semen was not included in our titer reduction assays. Our results show that vaginally applied carrageenan gel is not inactivated after 8–24 hours, but there is a significant loss of vaginal gel that would reduce the overall anti-infective capacity.
The authors would like to thank the study participants for their contribution of time, energy, and commitment to this effort. The authors would also like to acknowledge colleagues at the Chiang Rai Health Club, Thailand Ministry of Public Health, United States Centers for Disease Control and Prevention, and the Population Council who contributed to study implementation and Dr Victoria R. Polonis for the subtype-AE virus used in this study.
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