Tenofovir is a common anti-HIV drug that in the currently approved dose (300-mg tablet) suppresses HIV-1 replication in vivo
and in vitro
but is not reported to markedly affect other viruses (Balzarini et al., 1993
). Recently, however, it was reported that, in the microbicide CAPRISA 004 trial, topical vaginal administration of tenofovir significantly diminished the acquisition not only of HIV-1 but also of HSV-2. We hypothesized that the discrepancy between the earlier reported lack of significant antiherpetic activity and the CAPRISA 004 data is explained by the striking differences in drug concentrations between systemic and topical applications of tenofovir.
We demonstrated that the antiretroviral drug tenofovir is indeed endowed with a direct antiherpetic activity in a variety of experimental models at drug concentrations that are lower than that the median concentration achieved in cervico-vaginal fluid following the administration of 1% tenofovir gel and that were non-toxic for the exposed cells (Rohan et al., 2010
and data of this study). Indeed, tenofovir levels in the cervico-vaginal fluid were reported to be 18.6 mg/ml.hr measured over a 24 hr-time period (AUC24hr
) after topical administration and still ~ 100 μg/ml at 24 hr post drug exposure (Schwartz et al., Abstract LBPEC03, 5th IAS, Cape Town, South Africa, 19-22 July 2009). Instead, it was shown by Dumond et al. (2007)
that the steady state genital concentration of tenofovir was around 100 ng/ml (24 hrs) and peak concentrations of tenofovir reached around 500 ng/ml (6 hrs) after administration of tenofovir by oral route.
Several recent publications report lower concentrations of tenofovir detected in the female genital tract upon in vivo
application, with some of them equal to or even slightly higher than EC50
we determined in vitro
and ex vivo
. Our findings might explain why HSV-2 transmission prevention by tenofovir was not absolute but reached 51% (Abdool Karim, 2010
), although the extrapolation of active drug concentrations ex vivo
to the situation in vivo
has its limitation.
We observed tenofovir activity against both laboratory and clinical HSV-1 and HSV-2 isolates (wild-type and drug-resistant thymidine kinase-deficient virus strains) in: (i) HEL cell fibroblasts; (ii) primary macrophages and keratinocytes; (iii) organotypic epithelial raft cultures; (iv) human lymphoid and cervical tissues ex vivo
; and (v) HSV-1- and HSV-2-infected mice. The most pronounced antiherpetic activity of tenofovir was observed in macrophages. Although HSV targets in tissues are still poorly understood and it is not known whether macrophages are important HSV targets in vivo
, it seems that both HIV-1 and HSV can infect macrophages.
HIV-1 has been frequently recovered from genital herpes lesions in coinfected individuals (Schacker et al., 1998
). Cells of the monocyte/macrophage lineage reside in genital mucosal tissues and are thought to be reservoirs of HIV-1 in the genital tract (Lehner et al., 1991
; Spira et al., 1996
). Moreover, there is evidence that HSV infection can also stimulate macrophages in vitro
and can induce HIV-1 replication in these cells (Moriuchi et al., 2000
). The observed marked inhibitory activity of tenofovir against HSV-2 in M/M cultures is likely due to the low endogenous dNTP pools in this cell type (Perno et al., 1996
) and/or to low HSV-2 replication in this cell type. Low endogeneous dNTP pools give tenofovir a competitive advantage to interact with the herpetic DNA polymerase activity. Furthermore, we deciphered the molecular mechanism of the tenofovir anti-herpetic activity: tenofovir diphosphate, to which tenofovir converted in various human cell types efficiently inhibits HSV DNA polymerase. Concentrations of this compound in the cells were in the range of concentrations that inhibited HSV DNA polymerase in a cell-free system.
Thus, our findings provide a direct explanation for the dual anti-HIV/HSV activity reported for CAPRISA 004 microbicide trial (Abdool Karim et al., 2010
; Cates, 2010
). Indeed, in our experiments tenofovir suppressed HSV activity at concentrations of ~ 10-200 μg/ml, which are in the range of the drug concentrations reached in cervico-vaginal fluid upon application of 1% tenofovir gel (Schwartz et al., Abstract LBPEC03, 5th IAS, Cape Town, South Africa, 19-22 July 2009). Such tenofovir concentrations were not found to be toxic in our cell models, in agreement with previous findings that a 1% tenofovir gel does not affect either PBMCs or epithelial cells (Rohan et al., 2010
). Neither was any toxicity observed in the CAPRISA 004 trial. Shortly after the publication of the CAPRISA 004 results, oral Truvada, a combination of tenofovir disoproxil fumarate (TDF) and emtricitabine, was reported to provide a 44% reduction in the incidence of HIV in case of preexposure chemoprophylaxis in men who have sex with men (Grant et al., 2010
). In contrast to topical application, steady-state tenofovir concentrations in the genital tract following oral administration (300 mg/day) have been shown to be around 100 ng/ml (Dumond et al., 2007
While tenofovir concentrations generated during oral drug administration may be sufficient for an effective systemic inhibition of HIV infection, they are substantially lower than those necessary to inhibit the replication of herpesviruses. Accordingly, prevention of HSV-2 acquisition was not reported in this trial. Also, no epidemiological evidence has emerged of a concomitantly decreased incidence of HSV-2 infection in HIV-infected individuals treated with systemic (oral) tenofovir DF. In fact, it was very recently reported that oral tenofovir as part of combination antiretroviral therapy had no suppressive effect on HSV shedding in HIV/HSV-coinfected asymptomatic adults (Tan et al., 2011
). Also, another recent report showed that daily oral tenofovir DF (in co-formulation with FTC) did not reduce HSV-2 acquisition among high-risk men who have sex with men, likely due to TDF concentrations in the rectal or penile tissues being insufficient to decrease the acquisition of HSV-2 infection (Lama et al., Abstracts of the 18th
Conference on Retroviruses and Opportunistic Infections, Boston, Massachusetts, USA, 27 February-2 March 2011, # 1002).
Surprisingly, in contrast to the partial prevention of HIV acquisition upon topical intravaginal administration of a 1% tenofovir gel (Abdool Karim et al., 2010
) and upon oral drug administration in men who have sex with men (Grant et al., 2010
), the FEM-PrEP HIV prevention study with oral Truvada has been stopped due to inability to determine effectiveness (http://sciencespeaksblog.org/2011/04/18/fem-prep-hiv-prevention-study-halted-due-to-futility/
). No significant differences on the number of new HIV infections were observed between placebo and drug-treated individuals. Although the reasons for this apparent discrepancy have not been revealed yet, it should be clear that the oral route of tenofovir administration does not affect the rate of HSV-2 sexual transmission (Tan et al., 2011
), in contrast with the topical route of drug administration. This conclusion will be further tested in the ongoing VOICE clinical trial, which has both topical and systemic application arms.
Although we have consistently demonstrated the anti-herpetic activity of tenofovir in different systems, its potency is predictably lower than that of specific anti-herpetic drugs, like acyclovir. Indeed, tenofovir was designed to suppress HIV-1 rather than herpesviruses. However, when the drug was tested upon oral administration, the high concentrations that are achieved with topical application were not considered. With the CAPRISA 004 trial it became clear that these concentrations have now become clinically relevant and have been shown to suppress not only HIV-1 but HSV as well. Here, we showed that tenofovir affects HSV directly rather than through a complex indirect mechanism. Together, these findings argue that “marginal” antiviral activities of a variety of existing drugs should be revisited in the light of possibly missed antiviral activities in topical applications as new microbicides with dual or multiple antiviral properties. In this respect, it would be important to consider acyclovir prodrugs as potential microbicidal candidates. Indeed, acyclovir, which has traditionally been regarded only as a potent anti-herpesvirus inhibitor, has recently been shown to have dual antiviral properties: in lymphoid and cervico-vaginal tissues coinfected with herpesviruses, it efficiently inhibits HIV-1 as well (Lisco et al., 2008
Thus, in this respect, like tenofovir, acyclovir prodrugs can become potential dual-targeted microbicides. Importantly, it has been shown that prodrugs of phosphorylated acyclovir that bypass the requirement of the presence of herpesvirus for drug activation (phosphorylation) release the activated form of acyclovir intracellularly and are endowed with both anti-herpetic and anti-HIV activity (Derudas et al., 2009
; Vanpouille et al., 2010
). However, the findings on anti-viral activities of various compounds in ex vivo
models (even in ex vivo
human tissues that closely reflect ones in vivo
) have their limitations and should be verified in clinical trials.
In conclusion, our data provide a plausible explanation for the unexpected anti-herpetic activity of 1% tenofovir gel observed among treated African women participating in the CAPRISA 004 trial. Furthermore, our results provide specific considerations for designing new microbicides with a dual antiviral effect and indicate that topical creams rather than oral administration of anti-HIV compounds, in particular of tenofovir and its derivatives, may be efficient in preventing transmission of HIV-1 and its copathogens.