To enable an evaluation of the in vivo efficacy of prenylation inhibitors against HDV we established a novel mouse model of HDV that results in authentic HDV viremia. We then used this model to demonstrate the potent in vivo antiviral efficacy of two representative prenylation inhibitors. Indeed, despite a large intrahepatic burden of HDV, these compounds were able to completely clear HDV viremia to below the limit of detection. These results thus translate a previous in vitro observation (22
) into what we believe to be a relatively dramatic and clear first in vivo confirmation of the potential of this novel class of antiviral agents.
Using the delivery technique of hydrodynamic transfection and HBV-transgenic mice as recipients, we sought to recapitulate several key aspects of HDV superinfection of human chronic carriers of HBV. A relatively high intrahepatic burden of HDV was achieved, as reflected both in the total amount of HDV RNA (Figure a) and the percentage of infected cells (Figure e) within the livers of recipient mice. By also providing the replicating HDV with a source of its natural envelope protein, we were able to observe the production and release of authentic HDV virions into the serum (Figure ). Because HDV virion assembly in vitro is absolutely dependent on prenylation of the HDV large delta antigen protein, we hypothesized that administration of a specific inhibitor of the enzyme known to be responsible for delta antigen prenylation would result in abrogation of the HDV life cycle at the critical step of assembly and release into the serum. The presence of HDV viremia in our mouse model allowed us to test this hypothesis directly. When HDV-viremic mice were treated with either of two prenylation inhibitors at doses known to inhibit prenylation in vivo, rapid and efficient clearing of HDV from the serum was observed.
This model has several attractive features compared with other HDV animal models (34
). Because the mice are transgenic for HBV and therefore immunotolerant of these viral antigens, experimental HDV infection of these animals shares several important aspects with HDV superinfection of human chronic HBV carriers. As opposed to chimpanzees or woodchucks (34
), the highly inbred nature of the mouse can help avoid problems such as the wide spectrum of liver disease observed among animals receiving a common inoculum (35
). Our mouse model may prove particularly useful for studying host responses to viral replication in the setting of limited animal-to-animal genetic variability and, by crossing with other strains, specific genetic backgrounds. Because the mice are inoculated with cloned DNA, we have the ability to introduce HDV genomes with any desired mutations or genotype and thereby evaluate the effect of the latter on infection or pathogenesis. Finally, in contrast to xenotransplanted immunodeficient mice (40
), the relative technical simplicity and low cost of this new model, coupled with the small size of the host animal, is ideal for experiments involving precious compounds available in only limited quantities.
As hypothesized, inhibition of HDV viremia appeared to be at the prenylation-dependent step of virus assembly and release, since intrahepatic HDV RNA levels were comparable in mice treated with drug or vehicle control. The observed clearance of viremia was not simply a nonspecific hepatotoxic effect of the FTIs because ALT levels were also comparable among treatment groups. Rather, as observed in HDV in vitro cell culture systems (32
), it is a critical stage of the viral life cycle — the ability of replicated HDV RNA to be packaged and released in the form of progeny virions — that is specifically and dramatically disrupted by the prenylation-inhibiting compounds.
Our results support our initial hypothesis and demonstrate that prenylation inhibitors can indeed effectively inhibit HDV viremia. These results have obvious clinical relevance and importance for human HDV infections against which effective medical therapy has been lacking to date. In our mouse model, newly produced virions cannot infect new cells because the mouse is not a natural host for HDV. Presumably, mouse hepatocytes lack the receptor for HDV. In human HDV infections, however, the ability to break a critical step in the HDV life cycle, such as virus assembly and release, would be expected to have a major impact on the subsequent spread and course of HDV infection and associated liver disease. Our results now provide the experimental foundation for testing this hypothesis directly in a larger animal model whose hepatocytes are naturally permissive for HDV infection, such as the woodchuck (49
It is interesting to note that the antiviral strategy we have outlined represents a paradigm different from classical antiviral approaches, since we are not actually directly targeting a viral protein. Rather, the prenylation inhibitors are designed to deprive the virus access to a host function, namely farnesyltransferase activity. Thus, in contrast to traditional antiviral agents that bind a viral enzyme capable of mutating to develop resistance, it may prove to be particularly challenging for HDV to develop resistance to prenylation inhibition, especially because the relevant genetic loci are not under the virus’ control. Moreover, because the farnesyl moiety on the delta antigen may act as a specific ligand (50
), its function might not be readily substituted by another prenyl lipid such as geranylgeranyl.
Considering that farnesyltransferase is a host cell enzyme, it is surprising that FTIs are so well tolerated even in in vitro studies (51
). Perhaps this results from the existence of a family of prenyltransferase enzymes and the ability of different prenyltransferases to occasionally cross-prenylate substrates. It appears, however, that HDV is unable to benefit from either of these back-up mechanisms. It is exciting that orally available FTIs have been developed (29
) and have been used in human phase I/II trials (52
) with relative lack of toxicity.
Finally, our use of prenylation inhibitors as antiviral agents represents a prototype for an antiviral strategy that may be applicable to a wide variety of viruses that may also use prenylation in key aspects of their respective life cycles. These viruses include a collection of other medically important viruses (31
), as well as potential agents of bioterrorism.