April 1, 2014
The persistence of a latent form of HIV integrated into the genome of long-lived cells, particularly memory CD4 T cells, is considered the most significant barrier to curing the infection. A key element of cure research is the pursuit of strategies capable of inducing latent HIV to replicate, with the aim of causing the death of the latently infected cell either as a result of viral cytopathic effects or recognition and elimination by HIV-specific immune responses (or other targeting strategies). A number of candidate latency-reversing agents (LRAs) have emerged from studies, with the anticancer drugs HDAC inhibitors leading the pack. Two clinical trials of the HDAC inhibitor vorinostat have indicated that it is capable of inducing latent HIV to begin making viral RNA, but whether subsequent steps in the viral life cycle ensued -- the manufacture of HIV proteins and their assembly into infectious virions -- is not yet known. Two recently published studies set out to better understand the capabilities of the current crop of LRAs, including HDAC inhibitors, and report that they may not be up to the task, at least as single agents.
C. Korin Bullen and colleagues from the laboratory of Robert Siliciano at Johns Hopkins University tested the activity of a panel of LRAs including the HDAC inhibitors vorinostat, romidepsin, and panobinostat, along with three compounds with differing mechanisms: disulfiram (acetaldehyde dehydrogenase inhibitor), JQ1 (bromodomain inhibitor) and Bryostatin 1 (protein kinase C agonist). Results were compared to those obtained with PMA/ionomycin, which maximally induces replication of latent HIV by activating all CD4 T cells. A critical aspect of the study is that it was carried out using CD4 T cells sampled from HIV-positive individuals on ART; most prior research on LRAs has relied on laboratory models of HIV latency.
The researchers first employed an assay that measures viral outgrowth, and were able to demonstrate HIV production by CD4 T cells from 11 of 13 donors after stimulation with PMA/ionomycin. In contrast, no virus production was detected after exposure to any of the LRAs. Similarly, extracellular HIV messenger RNA could be detected in most cases after PMA/ionomycin stimulation, but in only one instance with a single LRA (Bryostatin 1). Lastly, HIV reactivation was assessed with an assay that quantifies intracellular viral messenger RNA; levels were detectable at baseline in 10 of 11 samples, and only increased significantly in response to Bryostatin 1 and PMA/ionomycin.
The overall conclusions drawn by the authors are fairly grim: "Our data demonstrate that none of the leading candidate nonT cellactivating LRAs tested significantly disrupted the latent reservoir ex vivo. The discordance between the effects of nonstimulating LRAs in in vitro models of HIV-1 latency and their effects ex vivo in resting CD4+ T cells from infected individuals on ART indicates that these models do not fully capture all mechanisms governing HIV-1 latency in vivo." However, when second author Gregory Laird presented the findings recently at CROI (see webcast), he added the information that combinations of LRAs have since shown signs of promise; both romidepsin plus Bryostatin 1 and panobinostat plus Bryostatin 1 induced HIV RNA production to levels similar to PMA/ionomycin. Although its not clear if Bryostatin 1 is safe enough to enter human trials for HIV, several modified analogs are in development.
The second paper, published in PNAS by Anthony Cillo and colleagues from John Mellors laboratory, compared the activity of vorinostrat and CD4 T cell activation (using ant-CD3 and anti-CD28 stimulation), also using resting CD4 T cells isolated from individuals on ART. In this case, the researchers evaluated the proportion of integrated HIV that showed a response to each intervention. CD4 T cell activation led to HIV RNA production by 1.5% of the integrated viruses, whereas the proportion for vorinostat was 0.079% (not significantly higher than the background levels absent any intervention). While the relatively small proportion of latent HIV induced by CD4 T cell activation is partly explained by the presence of defective viruses, the authors note the results echo another recent study in suggesting the presence of a substantial HIV reservoir refractory to any currently used in vitro stimulus. Whether these viruses can be activated in vivo is not known. The paper concludes: "Identifying strategies to increase fractional proviral expression without inducing global T-cell activation is critical to achieving progress toward a cure of HIV-1 infection."
Update 4/10/14: An hour or so after this post was completed, PLoS Pathogens published a study by Datsen George Wei and colleagues reporting that the HDAC inhibitor romidepsin is capable of inducing virion production by latently infected CD4 T cells sampled from individuals on ART (see figure 3 and table S2 in the original published study). Results are also reported for vorinostat, but those seem compatible with the Bullen and Cillo papers described above. I'm not sure what the explanation might be for the seeming conflict with the results reported by Bullen et al for romidepsin, although perhaps they might relate to differences in duration of in vitro exposure to the drug as the most consistent effects reported in the PLoS Pathogens paper appear to occur after six days whereas Bullen et al used 18 hours. The proportion of latent HIV induced to produce virus by romidepsin is not reported. Two clinical trials are assessing the activity of romidepsin in HIV-positive individuals (see TAG's clinical trials table) so hopefully clearer answers about its activity will be forthcoming.
Richard Jefferys is the coordinator of the Michael Palm HIV Basic Science, Vaccines & Prevention Project Weblog at the Treatment Action Group (TAG). The original blog post may be viewed here.
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