Readers of TAGline are asked to harken back to the duelling ganciclovir studies unveiled this past winter (see the March TAGline, vol. 3, issue 2). Where the Syntex study #1654 (now Syntex/Roche) declared a huge benefit to the its drug in preventing the onset of CMV retinitis, the community-based CPCRA 023 study found no benefit at all. These conflicting results have remained a favorite subject of controversy at the years large AIDS meetings, especially in light of the drug's exorbitant price ($25,000-$30,000 annually). Now that the final analyses of these two studies have recently been completed, clues to the puzzling discordance have begun to emerge.
Antagonism Between Oral Ganciclovir and ddI Suspected to Diminish Anti-CMV Effect
Although one study has reported ganciclovir to decrease ddI's antiretroviral effect, ganciclovir is generally known to increase blood levels of ddI when the two drugs are taken together. The literature on ddI's effect on the pharmacokinetics of ganciclovir, however, is quite limited. This is about to change. For when the CPCRA 023 team combed through their data in their oral ganciclovir CMV prophylaxis study, they uncovered a curious (and antagonistic) relationship between concomitant use of oral ganciclovir and ddI. In their study, participants who received placebo ganciclovir and were also taking ddI experienced a very low rate of CMV disease. (That in itself seems odd.) By contrast, study participants who were actually receiving ganciclovir and were also taking ddI experienced a very high rate (7.5-fold increased risk, p= 0.02) of CMV disease. (Odder still.) In fact, of the 63 individuals in the placebo arm who were also taking ddI, only one developed CMV disease (that comes out to 1.9 cases of CMV per 100 person years). But for the 269 individuals in the placebo arm who were not taking ddI, 34 developed CMV (18.9 per 100 person years). So the CPCRA 023 results need to be looked at with this unexpected drug-drug interaction in mind; when ddI-taking individuals are removed from the analysis, prophylactic use of oral ganciclovir reduced the risk of developing CMV disease by 38%, compared to placebo (p= 0.04). (The Syntex study #1654, you may recall, reported that oral ganciclovir reduced the risk of CMV by 50%.)
The interaction between oral ganciclovir and ddI has baffled many. "Does ddI have some as yet undiscerned prophylactic effect against CMV?" became the obvious question. The curious prophylactic results seen with ddI had not been noted before. In fact, when the Syntex #1654 study team performed a post hoc analysis of all the #1654 patients who were taking ddI, they did not find the ddI effect that the CPCRA 023 team had reported. For their part, the 023 team stands by their analysis, but study chair Carol Brosgart cautions that the results should be interpreted carefully and that the negative ddI and oral ganciclovir interaction should be investigated further. Some people are now joking about getting Bristol (manufacturer of ddI) to design a CMV prophylaxis study with ddI alone or maybe in combination with its recently rediscovered anti-infective lobucovir.
Using CMV DNA PCR to Individualize Prophylactic Strategies
An interesting feature of the Syntex #1654 study was the monitoring of CMV-DNA by new PCR techniques. Virology specimens were available for 619 of the study's 725 participants, and the results are fascinating. For all study participants who were CMV PCR positive at study entry (approximate 55% positivity rate in both arms), CMV disease developed in 43% of the placebo recipients and 26% of the oral ganciclovir recipients (p=0.017). Among the CMV PCR negative patients at study entry, CMV disease developed in 1% of the oral ganciclovir recipients and 14% of the placebo recipients (p= 0.00001).
Quantitatively, CMV-DNA PCR positive persons were stratified into three groups: fewer than 2,500 copies; 2,500-50,000 copies; and 50,000-150,000 copies. Study participants with fewer than 50,000 copies of virus benefitted from oral ganciclovir: 20% of the oral ganciclovir patients developed CMV disease vs. 40% of those on placebo, p= 0.004). For those with greater than 50,000 copies of CMV virus, there was no protective effect of oral ganciclovir compared to placebo. Of note, there were 10 patients with over 150,000 copies (all randomized to the oral ganciclovir arm) and they all went onto develop CMV disease.
This viral load sub-study tells us three things: 1) CMV-DNA PCR negative patients can benefit from oral ganciclovir; 2) If a person is CMV-DNA PCR positive and has a low viral load (less than 50,000 copies) s/he will still get some benefit with using oral ganciclovir; and 3) If a person has high CMV-DNA viral load (over 50,000 copies), oral ganciclovir won't help. Curiously, the only other CMV prophylaxis study to use PCR techniques to assess qualitatively the CMV-DNA status of participants was the NIAID's valacyclovir study (ACTG 204). In that study, exactly the opposite predictive parameters were reported; that is, CMV PCR-positive persons responded very well to prophylactic therapy with valacyclovir, while those PCR-negative showed little benefit to anti-CMV prophylaxis.
Still, the #1654 study results point to some obvious questions: For someone who is CMV-DNA PCR positive with a CMV-DNA copy number over 50,000, is it worth (both in terms of medical economics and quality of life) prophylaxing with oral ganciclovir? Similarly, in a CMV-DNA negative person, what is the value of prophylaxis with oral ganciclovir (at $30,000 a year) when the risk of developing CMV disease is only 14%?
In many ways, this viral load sub-study of #1654 acts as a natural history study which tells us a great deal about who will might develop CMV. For a long time we knew that a CD4 cell cut-off of 50 cells was not sufficiently sensitive. If we look at all patients at baseline (those with under 50 CD4+ cells), approximately 50% will be CMV PCR positive and 50% will be CMV PCR negative. Approximately 15% of all the negatives and 45% of all positives will develop CMV end-organ disease. If a patient has high CMV viral load, his or her risk of developing CMV end-organ disease is ostensibly greater. These data have also given rise to the prospect of treating a CMV PCR positive person with a short course of intravenous ganciclovir (to get him or her CMV PCR negative) followed by oral ganciclovir, or nothing at all.
For nearly a decade Jay Levy, the crotchety virologist at the University of California in San Francisco, has been trotting from research meeting to research meeting touting the ability of unidentified soluble factors produced by CD8+ lymphocytes to suppress HIV replication in vitro. Over the course of those ten years, Dr. Levy and his colleagues have reported on this CD8+ antiviral factor (CAF) activity in HIV-negative people who repeatedly had sex with people later found to be HIV-infected and in HIV-negative infants born to HIV-infected mothers.
Although the phenomenon Dr. Levy described was real and reproducible in other labs, the identity of these mysterious antiviral factors continued to elude poor Dr. Levy, and he began to develop a reputation as something of a crank on the scientific symposia circuit. At long last, Dr. Levy's tenacious belief in his molecules was vindicated in December of 1995 when a group of Italian researchers published its work isolating a set of substances that were being pumped out by CD8+ T-cells and were shown to be potent inhibitors of HIV. The Italian team identified these substances as three members of a subset of cytokines known as beta-chemokines. And, in typical scientific fashion, gave them the tongue tripping moniker MIP-1-alpha and MIP-1-beta (for "macrophage inflammatory protein") and RANTES (for "regulated upon activation normal T-cell expressed and secreted"). Even though Dr. Levy has virtually denounced the Italian team's work, claiming these three chemokines -- and dozens of other cytokines -- to be impostors, with the real identity of his CAF still unknown (It is still possible that Levy's CAF may indeed turn out to be some other compound. Experiments by Anthony Fauci's lab at the National Institute of Allergy and Infectious Diseases (NIAID) point to additional anti-HIV factors secreted by CD8+ cells.), the Italian team's stunning discovery has paved the way for a completely unprecedented quantum leap forward in our knowledge about HIV.
While this ever-changing scientific drama played out on the international research stage, back in New York City, Bill Paxton, a brilliant Scottish postdoctoral fellow in Richard Koup's lab at the Aaron Diamond AIDS Research Center, was slaving away at his project examining individuals who, while repeatedly exposed to HIV, managed to evade infection with the virus. Paxton had shown that CD4+ lymphocytes from these individuals were relatively resistant to infection with primary, macrophage tropic (non-syncytia inducing or "NSI") isolates of HIV. Once the Italian group had published its beta-chemokine work, Paxton decided to see if his exposed, uninfected (EU) cohort had high levels of these cytokines.
Of some two dozen EU screened, Paxton found two individuals with CD4+ cells highly resistant to macrophage tropic HIV turned out to have 5 times the normal concentration of RANTES and elevated levels of MIP-1-alpha and MIP-1-beta! He also found that variations in HIV's viral envelope (the same variations that governed viral tropism for macrophage/monocytes or T-cells) could determine whether or not a given strain of HIV could infect cells from these two extraordinary EU volunteers. After Paxton had shown that CD4 receptor levels on his subjects' T-cells were normal, he mused that the second receptor for HIV, the identity of which (at that time) was still unknown (although they knew it to be a 7 transmembrane receptor), might be involved in protecting these lymphocytes from infection. Paxton's work suggested that the beta-chemokines were involved in some way in protecting at least these two EU individuals from infection with macrophage-tropic HIV -- probably by interfering with virus entry via the putative second receptor.
Down the northeast corridor, NIAID researcher Ed Berger had just discovered that the second receptor needed by HIV in order to infect a cell for T-cell tropic (syncytium-inducing, or "SI") strains of HIV was a 7-transmembrane receptor which he called LESTR (for "lymphocyte expressed T-cell receptor"). The coincidence was uncanny, and the significance of Berger's work was not lost on the researchers at Aaron Diamond. In no time at all, Paxton and another post-doc in John Moore's lab, Tanya Dragic, demonstrated (along with Paul Maddon's group at Progenics Pharmaceuticals) that HIV-resistant CD4+ cells from their two exposed, uninfected individuals could not fuse with cells expressing the envelope protein of macrophage tropic HIV. What's more, the beta-chemokines could block fusion between normal CD4+ cells and these macrophage tropic HIV envelope-expressing cells. The same phenomena could not be duplicated with cells expressing envelope proteins of T-cell tropic viruses. All this suggested that the second receptor for macrophage tropic strains of HIV was a beta-chemokine receptor.
Dragic and her colleagues quickly tested a dozen chemokine receptors to see if they were capable of assisting viral entry into CD4+ cells, but to no avail. Then another clue popped up for our scientific sleuths in corrections submitted to a paper by Philip Murphy of NIAID. Murphy's paper showed that the CC-CKR5 beta chemokine receptor bound a unique set of ligands: RANTES, MIP-1-alpha and MIP-1-beta -- the very same trio that the Italian group had identified as potent HIV suppressor factors and that were being churned out by Paxton's two exposed, uninfected wonders. Murphy's lab had also sequenced the CC-CKR5 gene, but selfishly refused to publish the data, hoping to get a jump on the other labs working towards pinning down the second receptor for macrophage tropic HIV.
In another freak instance of good fortune, the sequence for CC-CKR5 was discovered by Marc Parmentier's lab at the Free University of Brussels and published in April which allowed the receptor to be cloned and tested by the team at Aaron Diamond. Within a few weeks, Dragic and Paxton (and four other groups) had shown that CC-CKR5 was indeed the second receptor for primary, macrophage tropic HIV strains. Never before in the relatively short history of the AIDS epidemic had a series of scientific breakthroughs of this magnitude occurred. Within six months, a cocktail of beta-chemokines was discovered to have the ability to suppress primary, macrophage tropic strains of HIV in culture and to contribute in some way to HIV immunity in some individuals. At the same time, the identity of the second receptors for both T-cell tropic and macrophage tropic strains of HIV were also finally revealed. And the discoveries kept on coming. Paxton and Rong Liu, another post-doc at Aaron Diamond, soon discovered that the inability of CD4+ cells from the two exposed, uninfected individuals to become infected with macrophage tropic HIV was due to a homozygous (inherited from both parents) mutation in the genes for their CC-CKR5 receptors. Mark Parmentier's group in Belgium soon confirmed this work, and as Paxton and Liu had shown, found that about 1% of Europeans are homozygous for the mutation while 15-20% of Europeans are heterozygous (carrying a mutated gene from one parent and a normal gene from the other) for the defect. Curiously, the mutation did not show up in Africans or South Americans.
In September, in one of the uglier episodes (and there are many) in this frenetic period of discovery, Stephen O'Brien of the National Cancer Institute took advantage of his role in processing cells from half a dozen cohort studies to conduct an analysis of the frequency of the mutation in individuals in these epidemiological studies and its association with protection from HIV. Before any of his researcher colleagues could get their hands on the samples, O'Brien rushed a paper to press -- even as he was sending cells out to two groups (Paxton and Koup at ADARC and Philip Murphy at NIAID) who were planning to conduct similar analyses.
Most people involved in the affair had expected that O'Brien would simply work in parallel with the other investigators. Instead, he purposefully delayed sending out samples (O'Brien denies this) until he slimily got his own work completed and published. In any case, O'Brien's analysis showed the presence of the CKR5 defect in 11% of Americans of European descent and in 1.7% of African Americans. In addition, O'Brien's study suggests that infected individuals heterozygous for the mutation progressed more slowly to AIDS than those individuals with normal CC-CKR5 genes. Finally, the natural ligand for Ed Berger's LESTR (also called "fusin" or "CXCR-4"), the second receptor for T-cell tropic HIV, was discovered in August to be yet another chemokine, stromal cell derived factor 1 (SDF-1). This discovery was made by two groups, one at the Dana Farber Cancer Institute in Boston and the other at the Institute Pasteur in France.
Even after all of this unfolds, the story is far from over. As TAGline goes to press, new papers are in press at Nature and several other publications that will expand on the story of beta-chemokines and HIV co-receptor saga. To say the very least, it has been a bountiful year for basic research on AIDS.
At the IX International Conference on AIDS, the Treatment Action Group (TAG) debuted its recent treatment publication, The Wasting Report: Current Issues in the Treatment and Research of HIV-Associated Wasting and Malnutrition. Written by TAG's Tim Horn and David Pieribone, The Wasting Report is the first activist publication to concentrate on issues surrounding wasting disease. Dr. Donald Kotler, one of the nation's leading experts on wasting (St. Luke's/Roosevelt Hospital and Medical Center, New York), was the main advisor for the project.
HIV wasting is a multifactorial complication of HIV disease, involving complex interactions among HIV, the opportunistic infections and malignancies, chronic activation of the immune system, gastrointestinal organ destruction and endocrine and immune system dysregulation. Much remains to be learned about the pathogenesis, diagnosis and treatment of HIV-associated wasting. Evidence accumulated over the years indicates that weight loss, and particularly loss of lean body mass leads to shortened survival in people with HIV disease. HIV infection often results in the overall dysregulation of the body's metabolism, where an abnormally high metabolic rate leads to excessive breakdown (catabolism) of body protein. This is especially evident during the course of an opportunistic infection.
There is currently no consensus for the diagnosis of patients presenting with HIV-associated weight loss. Some doctors recommend a conservative approach, with no invasive diagnostic techniques and routine use of standard anti-diarrheal medications. The problem of this anachronistic approach is that wasting symptoms, casually lumped together under the term "AIDS enteropathy," often turn out to be, upon proper diagnosis, treatable opportunistic infections. Some might remain untreatable, but proper diagnosis is essential in effective treatment and management of symptoms. The most commonly employed diagnostic techniques in HIV wasting do not adequately discriminate between loss of body weight in general and loss of lean body mass in particular. New assays, such as bio-electrical impedance analysis (BIA), may provide a clearer picture of lean body mass depletion.
Treating HIV-associated wasting conditions is critically dependent on proper diagnosis and an appropriate nutritional regimen. Treatments range from anti-emetics (anti-vomitting drugs) to appetite stimulants, anti-diarrheals, metabolic treatments such as growth hormone or anabolic steroids, to cytokine modulators. There is currently no standard-of-care treatment for many aspects of HIV-related wasting, and effective treatments for several as-yet untreatable infections, such as cryptosporidiosis and microsporidiosis, are urgently needed. TAG's complete set of recommendations for advancing the research, diagnosis and treatment of HIV-related wasting and malnutrition can be found within The Wasting Report. Those interested in obtaining a copy of The Wasting Report can call us at (212) 971-9022 or fax us at (212) 971-9019. The suggested donation (to cover the costs of printing and postage) is $10.00.