The ideal microbicide should retain its in vitro activity against HIV and other STIs in vivo without disrupting the integrity of the epithelial barrier, inducing inflammation or adversely interfering with innate immunity. In this phase 1 study, several findings associated with VivaGel® were of concern. These included increases in genital cytokines and T-cell subsets that were associated with clinical findings of epithelial disruption, and that have been associated with an increased risk for HIV acquisition in women.
Epithelial damage can result in the release of several cytokines and chemokines including IL-1β
, IL-6 and TNF, IL-8, and IL-10. 5, 10
In accordance with this, visible epithelia damage in the vagina and/or cervix observed in our cohort was associated with increases in IL-1β
, IL-10 and TNF. We also saw elevations in the Th2 cytokines, IL-4 and IL-13, which have also been implicated in tissue damage. 11, 12
In comparison, the findings associated with VivaGel®
compared to placebo also showed a mixed picture of cytokine alterations which included greater levels of IFN-γ
In looking at the trend of cytokine expression across the 4 visit dates, the difference between the VivaGel®
arm and placebo arm at day 7 and 14 was in part due to the fact that the women in the placebo arm had lower levels on these days than seen at baseline. Because of the protocol design which enrolled women between days 5 to 14 of their menstrual cycle, the Day 7 visit normally occurred mid-cycle and the Day 14 visit occurred during the luteal phase. Several studies have shown that there are significant differences in cytokine levels seen during the menstrual cycle 13
, with consistent changes seen in IL-6 and IL-8 levels. Shrier et al 14
found mid-cycle dips whereas Al-Harthi et al 15
found much lower levels of IL-6 and IL-8 in the luteal phase compared to the follicular phase. Not all studies showed such contrasts during the menstrual cycle. 15–17
The absence of mid-cycle and luteal cytokine dips in the VivaGel®
arm may be interpreted as altering the normal immune cycle, and hence detrimental. The impact of such variations on HIV susceptibility in vivo
is not clear.
Epithelial damage also can lead to lymphocyte extravasation which, in the case of HIV, may enhance infection. Myer et al 18
showed that visible evidence of cervical epithelial disruption increased the risk of HIV acquisition. After epithelial injury, CD69+/CD8+ and CCR5+/CD4+ T-cells would be normally found in the epithelial mucosa as part of the host defense.
Unfortunately, CD4+ T-cells are also target cells for HIV. 19
Interestingly, the number of CD4+ and CD8+ T-cells in our study were increased in those with evidence of epithelial damage but activated CD4+ T-cells (CD69+ and CCR5+) were decreased for reasons not understood. Studies with N-9 demonstrate the product was associated with an influx of CD4+ T cells. Although we did not observe increases in the sheer number of CD4+ or CD8+ T-cells with VivaGel®
, we did observe greater numbers of activated CD8+ T-cells in the VivaGel®
arm at Day 7 compared to placebo; however, this difference was not sustained at Day 14.
Except for N-9, few other microbicide studies have incorporated immune markers. Bollen et al
examined the effects of Carrageen, a relatively inert substance without an apparent effect on HIV acquisition. In their study, Carrageen, as well as the placebo gel caused a decrease in the levels of IL-8 and IL-6, suggesting that the gels may have caused some inhibition in the assays. Decreases in IL-8 levels have been observed with the use of several microbicides, including cellulose sulfate, Pro2000 and BufferGel. 21–23
Pro2000 and BufferGel were also associated with lower levels of IL-1β
and SLPI. Since the placebo in our trial was the same substance (Carbopol) used as the vehicle for VivaGel®
, the dampening observed in the placebo arm for many of the cytokines was not likely due to inhibition by the vehicle itself.
One of the strengths of this investigation was that participants were all enrolled and subsequently followed at similar times of their menstrual cycle. A second strength of the study is that women were screened for numerous STIs and were excluded if any of the tests were positive. Third, the close observation of the women allowed us to control for possible factors such as visible blood and bacterial vaginosis which may also influence immune markers. Lastly, women agreed to abstain from sexual intercourse one week prior to enrollment and throughout follow-up and very few women had objective evidence of semen exposure. 6
Limitations include our small sample size which was consistent with most phase 1 trials. Also, the clinical interpretation of these findings is limited since there are no human trials that have yet shown that these immune markers define absolute risks for female HIV acquisition, although results from CAPRISA-004 are forthcoming. 24
Rather, the evidence for risk of HIV acquisition related to altered innate and adaptive immunity in the female genital tract are based primarily on in vitro
and animal studies. 3–5
In addition, the study involved two different populations. The impact of race and ethnicity could not be studied in this small sample size. However, in a previous publication 9
, we found significant immune differences between the two groups at baseline prior to product exposure. Future studies should include the potential impact of race and ethnicity on the association of microbicide products on mucosal immune changes in the genital tract.
In summary, in this phase 1 clinical trial, markers associated with epithelial damage were elevated in the VivaGel® arm compared to the placebo arm after 7–14 days of twice daily product use. Most of the changes were reversed 7 days after discontinuation of the product. These findings need to be taken with a certain degree of caution due to their exploratory nature and small sample size. However, these findings and the observed increase in adverse events reported in the two phase 1 trials should be taken into consideration in planning any future clinical trials.