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Smoking Out the Enemy

Figure 1Hope was once high that, over time, antiretroviral therapy would rid patients of HIV-infected cells. Such hopes hinged on the presumption that these drugs could reach any and all HIV reservoirs.That's clearly not the case, as the title of a recent conference in the French West Indies attests: the 1st International Workshop on HIV Persistence during Therapy. "HIV persistently replicates, even in infected patients whose levels of plasma viremia have fallen below detectable levels while on

By | February 2, 2004

<p>Figure 1</p>

Hope was once high that, over time, antiretroviral therapy would rid patients of HIV-infected cells. Such hopes hinged on the presumption that these drugs could reach any and all HIV reservoirs.

That's clearly not the case, as the title of a recent conference in the French West Indies attests: the 1st International Workshop on HIV Persistence during Therapy. "HIV persistently replicates, even in infected patients whose levels of plasma viremia have fallen below detectable levels while on [medication]," wrote Tae-Wook Chun.1 Thus, take a patient off the now-standard cocktail of antiretroviral agents known as highly active anti-retroviral therapy (HAART), and viremia comes roaring back (see related Feature story, p. 16).

SHOCK AND KILL

To eliminate the hidden HIV reservoir, researchers have been investigating strategies to activate HIV transcription in latently infected cells, and to eliminate selectively those cells that harbor the virus. Dean Hamer of the National Institutes of Health calls the combination "shock and kill."

Shocking is not new. The best known example, Hamer says in an interview, comes from trials in the 1990s in which the T-cell growth factor interleukin-2 (IL-2) was given to AIDS patients. Some hoped that in addition to boosting the patient's T-cell count, IL-2 would flush HIV out of hiding, allowing the newly reconstituted immune system to deal with it.

Selective HIV-specific cytocide also has been around for a while. Several groups have been working on targeting immunotoxins to infected cells. These poisons generally have been coupled to a moiety such as an antibody or receptor fragment that can recognize a portion of the HIV envelope located on the cell surface, with the hope that only the infected cells will take up the poison.

It was logical to put together induction with specific killing of the infected cells, Hamer explains. Several groups have recently joined the two approaches, at least conceptually, although most projects still concentrate on one or the other.

INITIATING THE SHOCK

Most of the research into activating latent HIV reservoirs relies upon stimulating the cells that harbor them. For this purpose, clinical investigators such as Thomas Jefferson University's Roger Pomerantz, the National Institutes of Health's Anthony S. Fauci, and the University of Amsterdam's Jan Prins, have each used the anti-T-cell receptor antibody (OKT3) in combination with IL-2. Alain Lafeuillade's group at General Hospital in Toulon, France, has combined IL-2 with the myeloid cell activator interferon gamma (IFN-γ), to stimulate outgrowth from macrophages and monocytes. These studies found that stimulation led to a reduction of residual HIV-1 disease, but as with IL-2 alone, none eradicated the virus.2

In the lab, scientists are investigating these and other ways to coax HIV out of its hidden sanctuaries. In 1998 Fauci and Chun stimulated resting CD4+ T cells, derived from HIV patients, in vitro with IL-2 and the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). They found potent induction of viral replication, which the HAART cocktail had completely suppressed.

More recently, researchers have been looking into analogs of 12-tetradecanoylphorbol-13 acetate (PMA), a potent inducer of cellular activation through the protein kinase C pathway. Of these, prostratin, which paradoxically inhibits HIV replication but induces expression of latent HIV, seem to be receiving the most attention. Also, numerous presentations cited the synthetic phorbol ester at the HIV Persistence workshop.

At the workshop, Pomerantz explained that prostratin's ability to induce virus from specific subsets of patient blood cell pools compared well with different cellular activators, including IL-2, OKT3, an HIV-1-specific capsid protein, the lectin phytohemagglutinin (PHA), and a molecular relative of prostratin. Paul Driedger of PKC Pharmaceutical in Woburn, Mass., had a poster showing that prostratin does not prevent proliferation of cytotoxic (CD8+) T cells in a mixed lymphocyte reaction. And Marjan Hezareh from the AIDS Research Alliance in West Hollywood presented evidence that prostratin induces down-regulation of the CD4 and CXCR4cell-surface receptors, but not the CCR5.

But, PMA is not useful in a clinical setting because of toxicity, Hamer notes, and while prostratin and kin clearly have reduced side effects, they "are still rather toxic." Hamer and his collaborators have been synthesizing large libraries of PMA analogs and testing them for side effects such as downregulation of cellular receptors and upregulation of cytokines. "The compounds work ... at least in the test tube," he told the workshop. "They all have side effects, but it's possible to change the therapeutic window by changing the chemical structure."

A TARGETED WAKE UP

Researcher Olaf Kutsch takes a slightly different approach to inducing virus. His group at the University of Alabama is attempting to target receptors exclusively or predominantly expressed on cell types that may be latently infected but are not major receptors involved in immune stimulation. Such costimulatory receptors still should activate the cell, but not result in the cytokine-release syndrome that can follow stimulation of the T-cell receptor or by IL-2, for example. While they have published work using a macrophage model, which demonstrates that stimulating the CD40 receptor can reactivate virus,3 Kutsch says it may not suit the clinical setting because of side effects that others have observed. He and his collaborators' current work focuses on using the T-cell coreceptor CD28 molecule of T cells, which they found reactivates HIV at about 69% of the level of PMA in patient cells without causing T-cell anergy or apoptosis.

Other labs are taking the strategy of "reactivating the viral expression with HDAC [histone deacetylase] inhibitors, without activating the T cells," writes Dominique Demonte in an E-mail. The Free University of Brussels researcher and his collaborators used trichostatin A and sodium butyrate, in conjunction with tumor necrosis factor stimulation, to open the chromatin of an infected cell line and allow transcription from the HIV long terminal repeat (LTR) promoter.45 They presented in vivo data at the workshop but emphasized the results' preliminary nature. David Margolis' lab at the University of Texas Southwestern Medical Center at Dallas is similarly using pyrrole-imidazole polyamides as HDAC inhibitors to derepress quiescent HIV LTRs in patients' resting T cells.

<p>Figure 2</p>

VISUALIZING HIV-1 REACTION

To screen for potential HIV-1 reactivating molecules, latently HIV-1 infected reporter cell lines [J89GFP] are used that express GFP as a marker for HIV-1 expression. Shown are fluorescent and bright field photographs of unstimulated and antibody stimulated [CD3, CD28] J89GFP cells.

There is always the danger that liberating viruses from their hiding places will trigger a new round of infection. If they make it to the clinical setting, most of these agents will be used in conjunction with antiretroviral therapy, thus lessening that danger.

WORKING TOWARD THE KILL

Early on, researchers hoped that once the cytopathic HIV became reactivated, it would kill the cells that express it. "I think it's now quite clear that that won't work," Hamer notes. The reason, he says, is that HIV does not kill every type of cell that it infects. "For example, macrophages, which are a major reservoir, are not killed by HIV." Similarly, researchers hoped that after HIV is reactivated, the immune system would recognize and destroy these cells, which is also clearly not the case, Hamer continues. "So just shocking, I don't think, is going to work."

Hamer himself collaborates with several groups that are trying to find ways of eliminating reactivated cells, including Ed Berger at the NIH and Jefferson's Michael Root. Berger linked derivatives of the Pseudomonas exotoxin with molecules such as soluble CD4 or high-affinity antibody derivatives that bind to the HIV envelope protein. These have been used to target and kill infected cells in vitro with little collateral damage. Similarly, Root's lab has designed a protein called 5-Helix, which interacts with the HIV envelope, and combined it with Pseudomonas exotoxin.6

Investigators such as Jose Alcami Pertejo of the National Center for Biotechnology in Madrid have demonstrated in vivo the use of a fusion protein, combining portions of diphtheria toxin and CD4. The toxin was administered to immunodeficient mice, which had been reconstituted with cells from HIV-infected patients. The researchers found a significant reduction in viral load.

Shock and kill therapies may some day help those infected with HIV but not, most likely, without causing costly side effects. The goal would be to prevent infection in the first place, and that would require a preventative vaccine, says Bob Gallo, codiscoverer of HIV and the director of the Institute of Courtesy, David N. Levy, George M. Shaw, and Olaf Kutsch, University of Alabama Human Virology in Baltimore (see First Person, p. 11). "But that doesn't mean it will do any good for 50 million [currently] infected people."

Josh P. Roberts tcwriter@msn.com is a freelance writer in Minneapolis.

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