Chemokine Advances May Provide New HIV Therapies

STAGED INVASION: Understanding the role of coreceptors CCR5 and CXCR4 provides a clearer picture of AIDS disease progression, says NIAID's Edward Berger. TAKING AIM: Chemokine receptors provided a fixed target for therapy, while the mutating HIV virus is a moving target, remarks NCI's Stephen J. O'Brien. Increased knowledge about the doorways HIV uses to enter cells has opened up new avenues of inquiry for AIDS researchers. Over the past several years, scientists have discovered several chemokine receptors (the chemical entrances HIV needs to invade a cell). They have also identified proteins, called ligands, that latch onto these entrances and found genetic mutations that create immunity. This flurry of findings about the way HIV infiltrates cells has prompted researchers to look for therapies that stem the spread of the virus to healthy cells. Approaches include developing small molecules that would attach to these chemokine receptors, thus blocking infection; vaccinating with ligands to prevent or control infection; and genetically mimicking the effects of the mutation-induced immunity. Identification of the coreceptors CCR5 and CXCR4, both of which the virus needs in addition to the CD4 receptor, provided the keys for those strategies, explains Edward A. Berger, chief of the molecular structure section, Laboratory of Viral Diseases at the National Institute of Allergy and Infectious Diseases (NIAID). "It looks like the virus that initially gets in is CCR5-specific, and then later on the virus acquires the ability to use CXCR4." The CCR5-specific form of the virus, called macrophage-tropic (M-tropic), is asymptomatic, while the CXCR4-specific form, called T-cell-tropic (T-tropic) causes full-blown AIDS by attacking T cells. TINY BLOCKERS: Small molecules that bind to and block chemokine receptors provides one strategy for controlling the spread of HIV, notes Aaron Diamond's Nathaniel Landau. Chemokine receptors provide a tempting target for therapy because they represent an earlier point of intervention than that of protease inhibitors, explains Stephen J. O'Brien, chief of the Laboratory of Genomic Diversity at the National Cancer Institute (NCI). Protease inhibitors attack the virus once it has successfully infiltrated the cell. While protease inhibitors have achieved well-documented success assaulting HIV, the drugs have not been able to kill the virus as it mutates and develops resistance. "[Protease inhibitors] are going after a moving target," O'Brien explains. By contrast, chemokine receptor interventions aim for the entrances the virus uses to infiltrate the cell. "They don't mutate," O'Brien says of the receptors. "They're a fixed target." Finding small molecules to bind to, and thus block, chemokine receptors presents the best odds of success among the three therapies, according to Nathaniel R. Landau, staff investigator with the Aaron Diamond AIDS Research Center, which now is part of Rockefeller University in New York. He notes that the pharmaceutical industry has a good record of shutting down G-protein-coupled receptors-a broad family of receptors that includes CCR5 and CXCR4. Landau favors blocking CCR5 with a small molecule over blocking CXCR4, because studies of people without both copies of the gene that codes for CCR5 show they are highly resistant to HIV. These studies also indicate that people without CCR5 are healthy. "A drug directed against CCR5 might not have any side effects," he says. Cases of HIV-infected individuals seemingly resistant to the onset of AIDS fed the competitive fire of research teams searching for ways to thwart the disease. Jay Levy and colleagues at the University of California, San Francisco, found suppressor activity in T cells of patients who were infected but seemed to be resisting disease progression (C.M. Walker, Science, 234:1563-6, 1986). In 1995, a group led by Robert C. Gallo, then head of the National Cancer Institute's Laboratory of Tumor Cell Biology, identified three chemokine molecules they concluded caused similar suppressor activity-RANTES, MIP-1a, and MIP-1b (F. Cocchi et al., Science, 270:1811-5, 1995). Still, the researchers didn't know to what part of the cells these ligands bound. The next discovery didn't answer that question. In the spring of 1996, Edward A. Berger, chief of the molecular structure section, Laboratory of Viral Diseases at the National Institute of Allergy and Infectious Diseases (NIAID) and colleagues identified a coreceptor, CXCR4 (Y. Feng et al., Science, 381:667-73, 1996). However, that coreceptor served only as a door for the T-tropic virus, a form of the virus that attacks T cells, associated with a later stage of infection. It also was not affected by the three chemokines named by the Gallo group. This realization triggered a race for a second coreceptor. Michael Samson and Marc Parmentier of the Free University of Brussels and their collaborators isolated the gene for a receptor onto which RANTES, MIP-1a, and MIP-1b cling (M. Samson et al., Genomics, 26:522-6, 1996). A few months later, five separate groups reported within one week of each other that CCR5 was the coreceptor for the macrophage-tropic (M-tropic) version of HIV, the version of the virus that first infiltrates the cells. Then, a group at the Aaron Diamond AIDS Research Center, now part of Rockefeller University in New York, discovered two patients who, despite repeated exposure to the virus, remained uninfected. The researchers determined that a mutation that deleted both copies of the gene that codes for the coreceptor CCR5 caused their resistance (R. Liu et al., Cell, 86:366-77, 1996). Parmentier and colleagues reported the same mutation a week later. Several groups rushed to find the mutations in larger populations. A group led by Stephen J. O'Brien, chief of the Laboratory of Genomic Diversity at the National Cancer Institute (NCI), beat the Aaron Diamond group into publication using the same samples provided by the Multicenter AIDS Cohort Study, a collaboration funded by NIAID to gather HIV samples from around the United States (M. Dean et al., Science, 273:1856-62, 1996). O'Brien drew criticism from members of the Aaron Diamond group and others who expected to receive the samples from him immediately, but instead waited several weeks (J. Cohen, Science, 273:1798, 1996). The Aaron Diamond group corroborated the O'Brien study a month later (Y. Huang et al., Nature Medicine, 2:1240-3, 1996). Both analyses showed that patients with the two-copy deletion were indeed resistant, and that patients with only one intact copy experienced slower disease progression. Most recently, O'Brien's group reported a mutation in the gene that codes for SDF-1-a ligand that attaches to the CXCR4 coreceptor. Although the mechanism remains unknown, O'Brien hypothesizes that the mutation causes protection by triggering the overproduction of SDF-1 (C. Winkler et al., Science, 279:389-92, 1998). P.S. In the best-case scenario, a CCR5-specific drug delivered while the virus is M-tropic would prevent it from turning T-tropic. In the worst-case scenario, blocking the CCR5 receptor would force the virus to mutate to the T-tropic version or make it find other viable coreceptors. "It's like you have multiple entrance ways and only close one," Landau says. DOMINANT ROLE: Although many other coreceptors have been discovered in the past several years, CCR5 and CXCR4 remain the most important, according to experiments conducted by Aaron Diamond's Linqi Zhang. However, Linqi Zhang, Landau's colleague at the Aaron Diamond center, doubts the latter outcome. In a recent paper, he and collaborators tested 20 versions of the virus in vitro and found that other chemokine receptors, including CCR2b and CCR3, play only a minor role in infection despite variations in the strains (L. Zhang et al., AIDS Research and Human Retroviruses, 13:1357-66, 1997). He also presented an expanded version of the study at the Fifth Conference on Retroviruses and Opportunistic Infections in Chicago last month. The newer study used 137 HIV subtypes, including HIV-2 strains. The previous study included only HIV-1 subtypes. "Regardless of the huge diversity, they all used those two coreceptors," Zhang tells The Scientist. "That supports the dominant role of CCR5 and CXCR4 in HIV entry." POLYPEPTIDE APPROACH: University of Pennsylvania's Robert Doms demonstrated in vitro that a polypeptide could block infection by T-tropic strains of HIV. Several groups have demonstrated in vitro blocking of the CXCR4 coreceptor. A Japanese group reported the in vitro success of a peptide inhibitor derived from horseshoe crabs (T. Murakami et al., Journal of Experimental Medicine, 186:1389-93, 1997). In the same issue, another group, led by Benjamin J. Doranz and Robert W. Doms, professors of pathology and laboratory medicine at the University of Pennsylvania Medical Center, reported that the polypeptide ALX40-4C blocks infection by T-tropic strains of HIV (B.J. Doranz et al., Journal of Experimental Medicine, 186:1395-400, 1997). The polypeptide was tested earlier in clinical trials and judged to be unsuccessful because it failed to reduce viral loads. "It actually was in a clinical trial for a different reason," Doms explains, adding that the data from that trial did not show whether or not the viral load was limited to the M-tropic variety. Doms and colleagues are seeking permission to re-examine the samples in order to test them for both M-tropic and T-tropic strains. If the samples contain mostly M-tropic HIV, the compound could be on a fast track for approval, since it has already undergone Phase I and Phase II testing. "There's no toxicity associated with this compound," Doms says. However, that polypeptide isn't a true small molecule. Another group from the Aaron Diamond center recently reported the in vitro success of AMD3100 in blocking CXCR4, but not CCR5 (G.A. Donzella et al., Nature Medicine, 1:72-7, 1998). That compound is a small molecule, meaning the potential for success in the body could be higher than for the peptides. "One concern about peptides is that they are relatively quickly metabolized," says John P. Moore, senior author of the Aaron Diamond study; Doms acknowledges that concern. Unfortunately, AMD3100 has the opposite problem-poor uptake by the body. However, Moore argues that its success in vitro demonstrates that the small-molecule approach works-it's just a matter of finding the right compound. "That should encourage drug companies to do screenings of their libraries." QUANTITY AND QUALITY: Institute for Human Virology's Robert Gallo suspects that delivering modified chemokines in the proper dose could slow the spread of HIV. Another approach to blocking HIV from cells involves using both therapeutic and preventative vaccines made from altered versions of the chemokine receptors' natural ligands. Although RANTES, MIP-1a, and MIP-1b-three chemokine molecules believed to cause suppressor activity-inhibit the M-tropic virus in lymphocyte cells, none of those chemokines completely blocks infection in primary macrophage cultures. However, Robert C. Gallo, director of the Institute of Human Virology at the University of Maryland in Baltimore, thinks the approach is still feasible. "If you can get the chemokine delivered in the proper amount, you should block entry and contribute to therapy." His lab purified macrophage-derived chemokine (MDC) and determined that it bound to receptors on activated T cells (R. Pal et al., Science, 278:695-8, 1997). Experiments by his group suggest that MDC suppresses the infection of T cells by both M-tropic and T-tropic viruses. Another group created a modified version of RANTES (AOP-RANTES) and tested it on both macrophage and lymphocyte cells. The engineered chemokine bound to CCR5 receptors in higher concentrations than the three natural chemokines. AOP-RANTES also failed to cause inflammatory responses-an unwanted effect associated with high levels of the three natural chemokines (G. Simmons et al., Science, 276:276-9, 1997). Gallo says one problem with flooding the receptors with the three natural chemokines is the potential to trigger usage by other variants of HIV or to trigger rapid mutation. He hypothesizes that a cocktail of MDC and AOP-RANTES could provide an effective way to prevent HIV infection and reduce the mutation threat. He also notes that several groups, including one led by Thomas Lehner, a professor of immunology at Guy's Hospital in London, have experimentally correlated increased production of chemokines in monkeys vaccinated against SIV-the animal equivalent of HIV-with protection from infection. Those experiments show that, theoretically, a preventative vaccine based on chemokines is possible. O'Brien, however, doubts that approach will be effective. "You can't vaccinate against your own proteins," O'Brien says. The most successful vaccines recognize foreign invaders, he adds. Moore, too, has reservations about the chemokine approach, although he agrees that it's still an avenue worth pursuing. He questions how specific the chemokines are for CCR5. He also wonders about toxicity and bioavailability. "Chemokines in blood have a short half-life," Moore says. CORECEPTOR VECTOR: University of Pennsylvania's James Hoxie developed a coreceptor-based vector to target specific stages of the disease. Aaron Diamond AIDS Research Center http://www.adarc.org Institute of Human Virology, University of Maryland http://www.ihv.org National Institute of Allergy and Infectious Diseases http://www.niaid.nih.gov/research/Daids.htm Knowledge of the coreceptors' roles in infection prompted James A. Hoxie, a professor of medicine at the University of Pennsylvania Medical Center, and his colleagues to develop a way to potentially deliver gene therapy to cells specifically infected with HIV (M.J. Endres et al., Science, 278:1462-4, 1997). The researchers coated some retroviral vectors with CD4 and CCR5 receptors and others with CD4 and CXCR4 receptors. The vectors with CCR5 attached to the envelope proteins of cells infected with the M-tropic virus, while the vectors containing CXCR4 linked to the envelop proteins of cells infected with the T-tropic virus. Neither vector infected other cells. While the experiment demonstrates that vectors can target cells with different tropisms, Hoxie warns that the approach is a long way from being available in humans. Neither set of vectors carried or delivered therapeutic genetic material into the infected cells. "It's not a big leap into therapy," he cautions. However, the same approach could be used on other viruses. Landau agrees that treating HIV with gene therapy is problematic. "I'm not optimistic," he states. For gene therapy to be effective, an engineered gene would have to be delivered to a majority of the infected T cells. The virus replicates so quickly that this represents a difficult proposition, at best. Berger cautions that researchers should not get too optimistic about the possibility of combating HIV by focusing on chemokine receptors. "I don't think stopping it from getting into the body once it's already been established [is the solution]," he says. And other issues of disease progression still need clarification-like the impetus for the CXCR4 strain to emerge. That issue is especially important for researchers seeking to target the CCR5 receptors. Blocking CCR5 could hasten the mutation toward the more T-tropic strain. Berger also notes that successes in vitro do not necessarily mean triumphs in vivo. The virus could find other receptors to use or develop into a new strain. Treating people with proteins that their bodies already produce could have an adverse effect. Gallo, too, feels scientists should continue to attack the disease from all avenues. Those avenues include better protease inhibitors as well as chemokine approaches. He echoes many of his colleagues who say that the virus has continued to amaze and confound them with its abilities to change and adapt. "I feel it would be a terrible mistake to be lulled into sleep."

Chemokine Advances May Provide New HIV Therapies

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