Drugs directly suppressing HIV play a major role in containing AIDS epidemics. However, it is becoming clear that other viruses that infect the human body prior to or after HIV infection play a significant role in HIV transmission, pathogenesis and disease. Therefore, the development of drugs that target not only HIV directly but also affect HIV by suppressing coinfecting viruses may be a valid strategy for HIV therapy. Here, we describe such a drug that suppresses both HIV-1 and HSV-2, one of the viruses that is commonly associated with HIV-1 infection and which facilitates HIV-1 transmission and pathogenesis.
ACV was designed to suppress HHVs, in particular HSV-2 [9
]. Its potency and specificity against HSV-2 and some other HHVs are based primarily on the unique ability of HHV nucleoside kinases to phosphorylate ACV and thus enable its eventual conversion into the 5′-triphosphate form, which is an HHV DNA chain terminator. Non-herpetic viruses, including HIV, lack such nucleoside kinase activity, and therefore ACV is inactive against them. However, recently we found that phosphorylated ACV is capable of terminating not only the elongation of HHV DNA but also that of HIV-1 DNA [10
]. This occurs in tissues coinfected with HSV-2 (or with other HHVs capable of phosphorylating ACV) and HIV-1, but not in HHV-free systems [10
]. However, ACV would be inactive against HIV-1 in tissues infected with HSV variants with nonfunctional TK (ACV-resistant HSV), and in tissues in which HHVs are absent, not replicating, or which phosphorylate ACV inefficiently. Thus, the anti-HIV activity of ACV heavily depends on HHV nucleoside kinases.
Here, we describe masked phosphorylated ACV derivatives (ACV ProTides) that suppress HIV-1 replication independently of HHV nucleoside kinases. The ProTide strategy has been earlier successfully used in designing an abacavir-based drug against hepatitis B virus [19
], d4T and L-C-d4A-based agents against HIV-1 [20
], and a 4′-azidonucleoside-based drug against hepatitis C virus [23
] as well as in several cancer studies [24
Here, we used the ProTide strategy to deliver intracellularly phosphorylated ACV. Inside the cells the ProTides undergo ester hydrolysis followed by P-N cleavage resulting in a release of free ACV-MP that is further converted into ACV-TP, which terminates HIV-1 DNA elongation. In contrast to other NRTIs such as tenofovir and adefovir, which are “stable” acyclic purine nucleoside phosphonate analogues, phosphorylated ACV is the first known non-phosphonate acyclic nucleoside analogue that inhibits HIV-1 RT.
We evaluated ACV ProTide activity in two experimental models of HIV infection: single cell cultures (T-cell lines or PBMC) and ex vivo
human lymphoid and cervicovaginal tissues in which the gross cytoarchitecture and local microenvironment are preserved and where anti-HIV drugs have already been evaluated [17
]. In these tissues 1 μM ACV ProTides was sufficient to inhibit HIV-1 replication by 75 to 100% and the EC50
s were as low as 0.14 to 1μM. In cell lines (MT-4 and CEM), ACV ProTides suppressed HIV-1 replication with EC50
between 1.7 and 12μM while they ranged between 4.9 and 55μM in PBMC. The lower potency of ACV ProTides in PBMC and cell lines compared to tissues may be related to the level of cell activation and proliferation. Immortalized cell lines and PHA-stimulated PBMC have high concentrations of intracellular pyrimidine and purine nucleotides, in particular of dGTP [27
], which competes with ACV-TP for DNA chain incorporation by HIV-1 RT. However, since the critical events of HIV pathogenesis in vivo
occur in tissues, the system of human tissues ex vivo
that supports HIV production without exogenous activation or stimulation appears to be more adequate.
To suppress HIV-1 replication in tissues, ACV Pro-Tides do not need to be present permanently: despite removal of the drugs several days post infection, inhibition of HIV-1 replication was sustained. It is conceivable that the block of the initial infection or the maintenance of effective intracellular drug concentrations even after its removal from the culture medium could be responsible for sustained HIV inhibition.
HIV replication was significantly inhibited either by brief exposure of ACV ProTide followed by its removal, or if the treatment was delayed. Thus, on the time-scale of our experiments, ACV ProTide can be applied before or after HIV infection, or removed during the course of infection and still significantly suppress HIV-1 replication in human tissues ex vivo. Moreover, ACV ProTides appeared to be inhibitory to a variety of HIV-1 isolates, irrespective of viral subtype and coreceptor tropism.
Importantly, none of the tested ACV ProTides killed cells in tissues or cell lines. Flow-cytometry of tissue cells treated with ACV ProTides even at a concentration 10 times higher than the EC50
did not reveal depletion of CD4+
T cells, or of their subsets, naïve TCM
, and TEMRA
. Neither did we observe cell death in cell line cultures with the ACV ProTides at a concentration as high as 150μM. However, it cannot be excluded that prolonged application of these compounds in vivo
may reveal mitochondrial poisoning that often takes several months to materialize in the case of the most toxic NRTIs [28
]. In vitro
, the tested compounds although being non-toxic were cytostatic but at concentrations clearly higher than their EC50
. Furthermore, the cytostatic effect was reversible since after removal of the compound, cells proliferated to the same level than in untreated control. The cytostatic effect of the ACV Pro Tides was not observed in ex vivo
tissues, since presumably cell proliferation was negligible in this system [29
While ACV ProTides inhibit HIV, they also retain their antiherpetic activity. In coinfected lymphoid and cervicovaginal human tissues, ACV ProTides efficiently inhibited both viruses. Furthermore, they suppressed an ACV-resistant strain of HSV-2 both in cell lines [12
] and in ex vivo
human tissues. These findings clearly demonstrate that the activity of ACV Pro-Tides is entirely independent of the virus-induced thymidine kinase.
The dual inhibitory activity of ProTides is important, since such drugs may interrupt a vicious circle of mutual facilitation of HSV-2/HIV infections. In particular, HSV-2 infection results in an increased risk of HIV-1 acquisition and transmission because of HSV-induced disruption of genital mucosa, recruitment of activated cells, and increase of HIV-1 genital and plasma loads [30
]. In turn, HIV-1 infection increases the frequency of HSV-2 reactivations and its mucosal shedding [34
Strategies to control HSV-2 in HIV-1 prevention and care are now being tested in several clinical trials. As of now, two targeted clinical trials failed to demonstrate a protective effect of HSV-2 suppression on HIV-1 acquisition [35
]. However these trials were marked by low drug dosage, as well as by low adherence and behavioral disinhibition [37
]. On the other hand, in HSV-2-positive individuals who were already infected with HIV-1, suppression of HSV-2 with ACV or its prodrug valacyclovir resulted in HIV-1 load decrease in plasma, genital, rectal, and seminal compartments [41
]. Thus, the pronounced antiherpetic activity of ACV ProTides [12
], in combination with their anti-HIV activity, seems to be an advantage of these compounds that can be used in the framework of these new strategies.
In conclusion, the anti-HIV activity of ACV ProTides, in conjunction with their potent anti-herpetic activity, makes them prototypes of new dual-targeted antivirals. Such drugs allow the suppression of both HIV and HSV-2, a copathogen that significantly enhances HIV-1 sexual transmission and acquisition. These antivirals, structurally based on ACV and intracellularly delivered by the ProTide strategy, may become valuable components of future anti-HIV drug cocktails.