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Clues to Non-Progressors

Flurry of Discoveries in Multiple Labs Leads to Identification of HIV's Second Receptor -- And New Therapeutic Target

The Good, the Bad, the Ugly

November 1996

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.