The demonstration that HAART could lower plasma virus levels to below the limit of the detection raised hopes that eradication could be achieved with prolonged therapy [Perelson AS, et al. Nature. 1997 May 8;387(6629):188-91]. However in recent years, there has been little discussion of a cure for HIV infection. Shortly after the advent of HAART, several groups showed that the virus could persist in a latent form in resting memory CD4 cells even in patients who had no detectable viremia for prolonged periods of time as a result of HAART [Finzi D, et al. Science. 1997 Nov 14;278(5341):1295-300; Chun TW, et al. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):13193-7; Wong JK, et al. Science. 1997 Nov 14;278(5341):1291-5]. These latently infected cells arise when activated CD4 cells are infected. They then survive long enough to revert back to a resting state as long lived memory cells. In these cells, virus production ceases, and HIV persists in a latent form as a double-stranded viral DNA genome stably integrated into host cell DNA. In addition to this latent reservoir, there are also small numbers of free virus particles in the blood of patients on HAART. In patients with clinically undetectable viral loads, this low level viremia can be detected with special assays [Dornadula G, et al. JAMA. 1999 Nov 3;282(17):1627-32; Hermankova M, et al. JAMA. 2001 Jul 11;286(2):196-207; Nettles RE, et al. JAMA. 2005 Feb 16;293(7):817-29]. Residual low-level viremia is indicative of continued virus production despite effective HAART, perhaps reflecting release of virus from stable reservoirs such as that in resting CD4 cells. These findings have led most investigators in the field to conclude that eradication of the infection will not be possible without novel approaches that eliminate viral reservoirs.

Despite the prevailing pessimism, several groups have been directly attacking the problem of viral reservoirs, and a recent report in Lancet goes so far as to raise the possibility of a cure [Lehrman G, et al. Lancet. 2005 Aug 13-19;366(9485):549-55]. This study involved the use valproic acid to activate latent HIV in resting CD4 cells. Valproic acid is an antiepileptic drug also used in the treatment of bipolar illness and migraine. Its mechanism of action is not entirely clear but may involve effects on concentrations of the inhibitory neurotransmitter, gammaaminobutyric acid (GABA). However, valproic acid also inhibits histone deacetylase (HDAC), an enzyme involved in chromatin remodeling and the repression of gene expression in various systems. Some studies have suggested that HIV latency involves changes in chromatin structure that make the latent provirus inaccessible [Jordan A, et al. EMBO J. 2003 Apr 15;22(8):1868-77], and a previous in vitro study had suggested that inhibition of HDAC could stimulate release of virus from latently infected cells [Ylisastigui L, et al. AIDS. 2004 May 21;18(8):1101-8]. The authors therefore carried out a small proof of concept study to determine whether valproic acid could reduce the size of the resting CD4 cell reservoir in vivo. The underlying assumption of the study was that activation of latent HIV in a patient on HAART would lead to death of the infected cells without further spread of the virus.

Because few investigators have been willing to speculate on the possibility of a cure in recent years, this report received a great deal of attention in the lay press and stimulated many inquiries from curious patients. It is important, therefore, to put this work in perspective. While the search for ways to eliminate viral reservoirs is clearly essential to finding a cure, there are several reasons why speculation about a cure is premature.

First, it is not entirely clear that the results will hold up in a larger study carried out over a longer time frame. Measurements of the size of the latent reservoir are notoriously difficult because of the scarcity of latently infected cells. Assays for latently infected cells, originally developed at Johns Hopkins [Chun T-W, et al. Nature. 1997 May 8;387(6629):183-8], involve the purification of resting CD4 cells from peripheral blood, the stimulation of these cells in vitro with mitogens, and the subsequent amplification of virus released by the activation of latently infected cells. After 2-3 weeks, virus growth is detected by an ELISA assay. By carrying out the cultures in a limiting dilution format, it is possible to estimate the frequency of latently infected cells. Typically, the frequencies are less than 1 per million. The error inherent in frequency measurements of rare cells is substantial, typically on the order to ± 0.7 log₁₀ c/mL. Thus, changes of less than 1.0 log₁₀ c/mL may not be statistically significant. It is therefore important that the provocative results of Lehrman and colleagues be confirmed in a larger study with longer follow-up.

A second major issue involves the underlying rationale for using HDAC inhibitors. In principle, such an approach might be useful if latency were exclusively due to changes in chromatin structure that prevent expression of viral genes. However, the molecular mechanism of HIV latency, although not entirely clear, is likely to be multifactorial (for a review, see Lassen K, et al. Trends Mol Med. 2004 Nov;10(11):525-31). A recent study shows that in resting CD4 cells, HIV DNA is usually integrated into introns of cellular genes that are actively being transcribed [Han Y, et al. J Virol. 2004 Jun;78(12):6122-33]. This result suggests that the lack of HIV gene expression in latently infected cells is not an accessibility issue. Rather, the transcription of HIV genes may be reduced by interference from the surrounding host gene and by the absence in resting cells of activation-dependent host factors needed for efficient transcriptional initiation and elongation [Nabel G, et al. Nature. 1987 Apr 16-22;326(6114):711-3; Tong-Starksen SE, et al. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6845-9; Herrmann CH, et al. J Virol. 1998 Dec;72(12):9881-8]. In addition, there are posttranscriptional mechanisms that may prevent virus production even if some transcription of HIV genes occurs [Seshamma T, et al. Proc Natl Acad Sci U.S.A. 1992;89:10663]. Thus, latency is complex, and it is not clear that inhibition of HDAC alone will overcome other blocks to HIV production in resting CD4 cells.

A third problem is that partial reductions in the size of the latent reservoir are not clinically useful. Latently infected cells are rare, and the occasional reactivation of these cells contributes only a very small amount of the virus produced in an untreated patient. Thus the reservoir does not present a problem unless cure is the goal of treatment. In this case, partial reductions are clinically insignificant, because when therapy is stopped, viral replication increases rapidly and exponentially until the previous set point is reached [Davey RTJ, et al. Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):15109-14]. This exponential growth means that in principle,even a single latently infected could rekindle the infection. Because of the exponential nature of the viral rebound, reductions in the latent pool should be viewed on a log scale. If the size of the reservoir is 10⁶ cells [Chun T-W, et al. Nature. 1997 May 8;387(6629):183-8], a 75% reduction still leaves 2.5 x 10⁵ cells. Eradication would require a >99.9999% reduction in the reservoir. In previous studies in which immune activation by interleukin-2 was used to affect a dramatic reduction in the size of the latent reservoir, treatment interruption led to a very rapid rebound in viremia [Davey RTJ, et al. Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):15109-14].

A final caveat is that there may be other cellular and anatomic reservoirs for HIV [Blankson JN, et al. Annu Rev Med. 2002;53:557-93]. These reservoirs may involve different mechanisms of persistence not affected by HDAC inhibition.

The problem of viral persistence in latent reservoirs is a truly daunting one, and in the view of these authors, talk of a cure is premature. Research on strategies for eliminating viral reservoirs is clearly essential. Even if a cure cannot be achieved, understanding viral reservoirs can contribute to the management of HIV infection. One critical issue is the ability of the latent reservoir in resting CD4 cells to store drug-resistant virus [Persaud D, et al. J Clin Invest. 2000 Apr;105(7):995-1003; Ruff CT, et al. J Virol. 2002 Sep;76(18):9481-92]. Any viral species that has circulated at high levels for a significant amount of time (weeks) has the potential to become archived in the reservoir, thereby permanently limiting treatment options. Another example involves the low level viremia that continues in patients on HAART who have undetectable viral loads. Recent studies of this low level viremia suggest that it represents release of virus from stable reservoirs without further replication or evolution [Kieffer TL, et al. J Infect Dis. 2004 Apr 15;189(8):1452-65]. This is true even in patients who experience “blips” [Nettles RE, et al. JAMA. 2005 Feb 16;293(7):817-29]. Thus, even if approaches for eliminating the reservoir cannot be developed in the near future, some comfort can be taken from the fact that HAART appears capable of fully arresting viral evolution in adherent patients. With optimal treatment, patients should be able to expect a normal life span.

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