This study demonstrates that entecavir is a potent but partial inhibitor of HIV replication in vitro and in vivo and that in some patients it can select for viruses bearing the M184V mutation, which confers high level resistance to entecavir and to the widely used anti-HIV drugs lamivudine and emtricitabine. These conclusions are supported by the striking temporal association of entecavir therapy with 1 log reductions in HIV RNA as well as in vitro studies showing inhibition of HIV infectivity at clinically relevant concentrations. In addition, one patient clearly demonstrated selection of M184V variants in vivo while on entecavir monotherapy and in vitro data demonstrate reduced activity of entecavir on the M184V variants.
These data have important implications for treatment of hepatitis B in HIV-infected patients. Current guidelines recommend entecavir as the first line treatment in HIV-HBV co-infected persons who do not require anti-HIV therapy (8
). Since this recommendation is predicated on the assumption that entecavir does not have activity against HIV, our data indicate that this recommendation should be reconsidered, especially since entecavir can select for the M184V mutation in some patients.
Our data contradict a previous report that entecavir does not have anti-HIV activity at clinically relevant concentrations (1
). That report used an assay described in 1989 by Weislow et al., which relies on cytopathic effects resulting from infection (21
). We believe the quantitative infectivity assay we used has several advantages over previous assays that allow it to detect subtle anti-HIV effects such as that shown by entecavir. First, the assay used here allows a more direct measure of drug inhibition of early steps in HIV replication from virus attachment through virus gene expression than older assays that used cell death as a surrogate measure of infection. Second, since the assay relies on a single round of infection, it allows rapid and precise quantification of individual infection events without the complications introduced by multiple rounds of infection in an extended culture. A third advantage is that drug inhibition of HIV replication is measured in primary CD4+
T lymphoblasts, which are the in vivo
target cells of HIV, rather than in transformed cell lines, which may metabolize entecavir differently.
Further work is needed to understand why the M184V mutation clearly emerged in one of the two patients who was studied in detail. It is unlikely that this patient was taking lamivudine since it was not being prescribed to him at the time or in the recent past and since complete emergence of this mutation from lamivudine occurs within weeks (22
). Rather, the appearance of the M184V mutation likely reflects the fact that it abolishes the anti-HIV activity of entecavir (). Interestingly, selection of the M184V mutation in HIV RT could be anticipated since structural models show that the M184V corresponds to the HBV pol
mutation, rtM204V (23
), which decreases HBV susceptibility to entecavir (24
). In both cases, the targeted methionine is in the YMDD motif at the active site.
The failure of the M184V mutation in HIV RT to become dominant in Patient #3 may reflect the fact that this mutation has a well known negative effect on viral fitness (25
). In some patients, this reduction in fitness may outweigh the modest benefit conferred by entecavir resistance since entecavir only partially inhibits HIV replication. Thus in some patients, selection for M184V may not occur or may occur very slowly.
It is also unknown whether the M184V variants that appeared in Patient #1 following entecavir treatment were initially generated by exposure to a lamivudine-containing HAART regimen several years earlier. Phylogenetic analysis suggests that the M184V variants evolved recently, most likely as a result of entecavir treatment. The appearance of M184V could also reflect emergence of an archived variant from the latent reservoir (26
), but in either case there is clear evidence that entecavir selected for this variant in patient #1.
Several other questions are also raised by this study. One question is whether control of HBV replication lead to diminished lymphocyte activation or alterations in cytokine release that affected HIV replication. Although this is theoretically possible, treatment of hepatitis B with adefovir dipivoxil in HIV-infected individuals does not support this hypothesis since the patients had stable HIV RNA levels despite declines in HBV DNA (27
). Although our data demonstrate that entecavir affects HIV replication, we cannot rule out that such secondary effects may have contributed to the decline in HIV RNA. Other questions include why the anti-HIV activity of entecavir plateaus at low nanomolar concentrations, whether modifications of the compound could overcome the plateau effect, whether entecavir could play a clinically meaningful role as an antiretroviral agent for HIV, and whether entecavir selects for other HIV drug-resistant mutants. Lastly, our data raise the question of the generality of the observations. We are aware of nine HIV-HBV co-infected patients who received entecavir monotherapy (three of whom are included in this report), and for all nine of them, the same simultaneous decline in HIV RNA was apparent.
In summary, entecavir, at doses used to treat chronic hepatitis B, is a potent partial inhibitor of HIV replication and can select for the M184V resistance mutation in HIV RT. Since the full extent of HIV RT mutations selected by entecavir monotherapy is not known, caution should be used in treating chronic hepatitis B with entecavir in HIV-infected patients not on fully suppressive antiretroviral regimens. Furthermore, these data underscore the importance of careful study of agents with potential for anti-HIV activity prior to licensure.