We evaluated the efficacy of gel containing TFV alone or in combination with FTC in preventing vaginal SHIV infection by using a repeat challenge macaque model that resembles human transmission. We demonstrate that topical application of a gel containing 1% TFV or a combination of 1% TFV and 5% FTC into the vaginal vault 30 min before virus exposure completely protected macaques against SHIV transmission. This suggests that the use of a 1% TFV gel close to the time of coitus may be sufficient to block transmission, without the need for application on noncoital days or the use of gels with drug combinations.
In our studies, we used female pig-tailed macaques (M. nemestrina
), which have a menstrual cycle similar to that of humans, with a mean cycle length of 32.8 days (5
). Pig-tailed macaques also have anatomy, pH, and bacterial microflora similar to those of humans (35
). Therefore, we were able to measure protection under normal cycling conditions, as macaques were challenged vaginally twice a week for up to 20 challenges or 10 weeks without the use of menstrual cycle synchronization or progesterone treatment, which can alter the vaginal environment and the susceptibility to infection (29
). The demonstration of high efficacy with repeated application of gels prior to exposure is reassuring because it does not indicate damage to the vaginal mucosa by the drugs or the gel formulation. Repeated use of nonoxynol-9 has led to cumulative mucosal toxicity, which may have neutralized the protective effect of this product or even increased virus transmission in humans (42
Previous macaque studies of systemic antiretroviral prophylaxis have not shown a high level of protection with only TFV or FTC despite daily treatment, indicating that regimens combining TFV and FTC were required to improve efficacy (10
). These findings and similar results from monotherapy of infected persons suggested that drug combinations were likely needed for highly effective topical prophylaxis (11
). Our findings that 1% TFV was sufficient to completely block transmission in this model are important and may be explained by the ability of the gel to deliver higher concentrations of drugs to vaginal tissues than are delivered via oral dosing. In addition, the twice-weekly dosing and the long intracellular half-life of TFV may both have helped in sustaining high and durable antiviral activity in vaginal tissues. Data comparing vaginal tissue drug levels in humans after oral or topical dosing will be highly informative (26
). Despite the low potency of TFV (IC50
of ~2 μM) (49
), this gel product was more effective in macaques than were gels containing similar concentrations of more-potent drugs (IC50
s of 1 to 20 nM) (43
). Therefore, in addition to potency and concentration in the gel formulation, other drug characteristics, like long intracellular persistence, may be critical to efficacy.
Our gel formulation allowed for rapid drug absorption, as both TFV and FTC were frequently detected in plasma 30 min after application. This may have contributed to the high protection by effectively blocking early virus infection that can occur within the first hours after virus challenge (6
). While both TFV and FTC were detected in the blood plasma of all animals, the generally higher detection frequencies and levels of FTC may be due to the fivefold-higher concentration of this drug than of TFV. With extensive sampling for plasma drug levels following 20 gel applications in each macaque, we also assessed macaque-specific variability in drug levels 30 min after vaginal gel exposure. Based on findings with six macaques who received one rectal gel application and a single virus challenge, Cranage et al. found a positive association between the degree of protection and the concentration of TFV in plasma 15 min after gel use (8
). We were unable to find a similar association between protection and plasma drug levels following vaginal gel application, since all animals were protected despite low levels of drug absorption. These data likely underscore the difficulty of identifying surrogate markers of protection from vaginal transmission based on plasma drug levels close to gel application.
Our model also provides an opportunity to examine immunologic priming in protected macaques that may have resulted from repeated virus exposures. Sequestration of virions by mucosal dendritic cells leading to cross-presentation of viral antigens may contribute to the development of such responses (17
). We observed SHIV-specific T-cell ELISPOT assay reactivity in only 2 of 12 protected macaques, while Cranage et al. reported more-frequent Gag-specific IFN-γ-secreting T cells in four of seven macaques protected from a single rectal exposure to simian immunodeficiency virus (8
). These responses may be either beneficial, because they reduce susceptibility to infection, or harmful, because they indicate T-cell activation and thus a higher susceptibility of target cells in the mucosa (16
). To help clarify the impact of observed T-cell responses on susceptibility to infection, we rechallenged both of our ELISPOT assay-positive macaques with repeated low doses of SHIV in the presence of the placebo gel and found no evidence of resistance to infection. Peak viral loads were similar for naïve and rechallenged macaques. A lack of resistance to infection was also observed for macaques protected from repeated rectal SHIV challenges by preexposure prophylaxis, although T-cell responses were not evaluated for these macaques (10
Our findings are in agreement with those of Cranage et al., who showed that a similar formulation of 1% TFV gel applied rectally up to 2 h before a single high-dose rectal challenge provided protection from infection with SIVmac251/32H in six of nine macaques (67%) (8
). Differences between the two studies may be related to the use of distinct macaque models with different stringencies or may reflect biological factors, such as the need for more-potent gels to protect a larger colorectal mucosal surface against a more efficient mode of transmission (40
). Further studies are needed to determine whether gels with combination drugs or higher TFV concentrations can enhance efficacy.
Our study is subject to several limitations. First, all viruses were inoculated in the absence of semen or semen-derived factors shown to enhance HIV infection in vitro (32
). Viruses were also inoculated on apparently intact mucosa without trauma, concurrent genital ulcers, bacterial vaginosis, or other conditions which can increase the risk of HIV acquisition by increasing the number of susceptible cells in mucosal tissues or enhance systemic dissemination of virus or virus-infected cells (3
). The availability of macaque models with chemically induced transient ulcers of the lower female reproductive tract would provide an important tool to assess efficacy on nonintact mucosa (48
). Second, since the contributions of cell-associated virus versus cell-free virus to establish infection in humans remain unclear, it may be prudent to confirm gel efficacy by using vaginal models of cell-associated transmission (19
). TFV and FTC are expected to be active against cell-associated infection, since the mode of action for RT inhibitors occurs intracellularly (19
). Third, human trials currently assess the safety and efficacy of 40 mg TFV present in 4 ml of 1% TFV gel. We decreased the volume of gel tested to 3 ml (corresponding to a lower dose of 30 mg TFV) to accommodate the smaller vaginal cavity in macaques. Although even smaller volumes of gel can be evaluated with monkeys to better mimic gel distribution in the human vagina, efficacy data from such studies would require cautious interpretation. A reduction in gel volume would disproportionately decrease the applied TFV dose and reduce drug exposure. Additionally, macaques clear TFV more rapidly than humans, further reducing drug exposure (10
Since all macaques in the TFV and TFV-FTC arms were fully protected from infection, drug resistance emergence in breakthrough infections could not be addressed in our study. Drug resistance concerns are important especially if gel products are not highly protective and include drugs that are used widely for treatment, as is the case with TFV and FTC evaluated in this study. However, whether a breakthrough infection would have resulted in the selection of drug-resistant virus is not known but may be less likely than expected because gel dosing results in lower levels of systemic drug exposure than oral dosing, which may not be sufficient for drug resistance selection. Therefore, because of differences in systemic drug exposure between oral and topical dosing with single drugs, a previous experience of drug resistance emergence from daily or intermittent monotherapy may be less relevant. Nevertheless, drug resistance emergence should be studied carefully with appropriate monkey models and current human trials by examining virus from plasma, rectum, and vagina.
In conclusion, we demonstrate that topical application of a gel containing 1% TFV or 1% TFV and 5% FTC 30 min before virus exposure completely protected macaques against numerous SHIV transmissions. Data from this model suggest that coital use of a 1% TFV gel may be sufficient to block HIV-1 transmission in humans, without the need for daily application or the use of gels containing drug combinations. This study highlights the high efficacy of this prevention strategy, supports clinical trials with humans, and informs the trial design by identifying a potentially highly effective modality.