At a symposium on cure research held prior to the International AIDS Conference in 2012, Robert Siliciano presented new data suggesting that the amount of replication-competent latent HIV that persists in the face of antiretroviral therapy (ART) may be greater than previously appreciated (the video and powerpoint from Siliciano’s talk are available on the symposium website). This research, led by Ya-Chi Ho from Siliciano’s laboratory at Johns Hopkins University, has now been published in the journal Cell. The paper expands on the originally reported findings, and concludes that the latent HIV reservoir may be at least 60-fold larger than prior estimates.
The genesis of the research was the well-documented discrepancy between the results of two different assays that are used to measure the HIV reservoir. The virus culture assay is a relatively complex and cumbersome approach that is designed to assess how much replication-competent latent HIV persists in the resting memory CD4 T cells of individuals on ART. It was this assay that Diana Finzi from Siliciano’s laboratory first used to document the existence of the latent HIV reservoir in the mid-1990s (showing that the reservoir size is typically around 1 per million resting CD4 T cells). Another much simpler assay involves the use of polymerase chain reaction (PCR) technology to measure HIV DNA levels. Studies have consistently found that when virus culture and HIV DNA results are compared, there is a sizable difference, with a recent careful analysis published in PLoS Pathogens documenting that HIV DNA levels average around 300-fold higher. The most commonly cited explanation for this discrepancy is the existence of large numbers of defective, replication-incompetent HIV viruses that are picked up by DNA tests but not by virus culture. Ya-Chi Ho’s aim was to closely scrutinize whether this explanation is correct.
The virus culture assay works by isolating resting memory CD4 T cells from individuals on suppressive ART and stimulating essentially all of them to divide with a mitogen (PHA). The stimulation causes most latent HIV present in the cells to replicate, leading to production of the HIV p24 protein, which can then be measured. For the purposes of this study, Ho focused on the CD4 T cells that contained integrated HIV DNA (HIV provirus) that was not induced to replicate by PHA stimulation. These “noninduced” viruses were analyzed in detail. As had been assumed, the majority of the noninduced viruses were found to have mutations that rendered them unable to replicate. But a substantial subset (25 out of 213 studied, or 11.7%) appeared intact. Further testing revealed that these viruses were replication-competent and did not appear compromised in any way; they could replicate just as well as laboratory HIV isolates and viruses from the same individuals that were induced after PHA stimulation.
In the last part of the study, Ya-Chi Ho evaluated whether latent HIV that was not induced by one round of PHA stimulation in the virus culture assay could replicate after a second round. The results showed that about a quarter of the resting CD4 T cells that did not produce HIV after the first PHA stimulation did produce viruses the second time around.
These findings led to the calculation that the replication-competent HIV reservoir is at least 60-fold larger than previously thought. Furthermore, the results of the experiment in which PHA stimulation was repeated suggest that chance plays a role in dictating whether or not a given latent HIV provirus will be induced to replicate after a latently infected CD4 T cell is activated. This chance (or stochastic) element could prove to be an obstacle for strategies that aim to cure HIV infection by inducing latent viruses to replicate so that infected cells can be targeted for elimination (the so-called “shock and kill” or "kick and kill" approach), because it implies that even repeated shocks might leave some latent HIV unmoved. In an accompanying commentary, Ariel and Leor Weinberger discuss this potentially stochastic aspect of HIV latency in greater detail.
The most immediate and serious implications in terms of clinical research may be for trials of HDAC inhibitors as antilatency drugs. There was a great deal of hooplah when results from the first human trial of the HDAC inhibitor vorinostat were published, indicating that some latent HIV could be induced to express viral RNA by the approach. But subsequent laboratory work has shown that HDAC inhibitors only affect a tiny fraction of the latent HIV that is induced by PHA stimulation, and now it appears that when all potentially inducible HIV is considered, the proportion of viruses that will respond to HDAC inhibition is even smaller. It remains to be seen if this will lead to a reevaluation of proposed trials of additional HDAC inhibitors, such as the phase I trial of romidepsin planned by the AIDS Clinical Trials Group and the combination study of romidepsin and a therapeutic HIV vaccine planned by the biotech company Bionor Immuno.
The publication of Ya-Chi Ho's paper has prompted a number of pessimistically tinged media stories (with at least one article overstating the results as a “major setback” for efforts to cure HIV). But there are some caveats: It is not yet known for certain that the noninduced viruses described in the study can be induced to replicate in the human body (although there is reason to suspect that they could be). Also, as the authors note, full replication of the virus may not be necessary in order for infected cells to be recognized and eliminated by the immune system; coaxing latent HIV to make some viral proteins might be sufficient. Additionally, not all proposed approaches to curing HIV are dependent on complete induction and elimination of the reservoir. Some—particularly gene therapies—are aiming for containment instead.
Note: Although the papers are not listed as open access, clicking on the PDF link next to the abstracts currently appears to take you directly to the full PDF files.
Cell, Volume 155, Issue 3, 540-551, 24 October 2013
Ya-Chi Ho, Liang Shan, Nina N. Hosmane, Jeffrey Wang, Sarah B. Laskey, Daniel I.S. Rosenbloom, Jun Lai, Joel N. Blankson, Janet D. Siliciano, Robert F. Siliciano
Antiretroviral therapy fails to cure HIV-1 infection because latent proviruses persist in resting CD4+ T cells. T cell activation reverses latency, but <1% of proviruses are induced to release infectious virus after maximum in vitro activation. The noninduced proviruses are generally considered defective but have not been characterized. Analysis of 213 noninduced proviral clones from treated patients showed 88.3% with identifiable defects but 11.7% with intact genomes and normal long terminal repeat (LTR) function. Using direct sequencing and genome synthesis, we reconstructed full-length intact noninduced proviral clones and demonstrated growth kinetics comparable to reconstructed induced proviruses from the same patients. Noninduced proviruses have unmethylated promoters and are integrated into active transcription units. Thus, it cannot be excluded that they may become activated in vivo. The identification of replication-competent noninduced proviruses indicates that the size of the latent reservoir—and, hence, the barrier to cure—may be up to 60-fold greater than previously estimated.
Cell, Volume 155, Issue 3, 497-499, 24 October 2013
Ariel D. Weinberger, Leor S. Weinberger
Classic studies proposed that stochastic variability (“noise”) can drive biological fate switching, enhancing evolutionary success. Now, Ho et al. report that HIV’s reactivation from dormant (latently infected) patient cells—the major barrier to an HIV cure—is inherently stochastic. Eradicating an incompletely inducible (probabilistic) viral phenotype will require inventive approaches.