The Emerging Race to Cure HIV Infections
Timothy Ray Brown's startling fate has pushed to the front a daunting research challenge that long seemed a fool's errand
The apparent cure of Timothy Ray Brown (left) has given momentum to novel interventions like the gene therapy that Matt Sharp (right) received.
CREDIT: BLOOMBERG/GETTY IMAGES
In May 1995, after learning that a former partner had become infected with HIV, Timothy Ray Brown, then 29, decided he should take the test himself. Brown, an American living in Berlin and working as a translator, felt perfectly healthy, had not had sex with his former partner in years, and 5 years earlier had tested negative. “It was a bit of a shock,” Brown says, when he found he was infected.
At the time, HIV was seen as a death sentence. AZT and two other antiretroviral drugs (ARVs) had come to market, but the pills typically delayed death from AIDS for only a few years. The damage to Brown's immune system wasn't yet life-threatening: His CD4 count, which refers to the white blood cells that HIV destroys, had dropped to under 400 cells per microliter. (Normal is 600 to 1200.) But his doctor recommended immediate treatment, and Brown started taking AZT.
Within a year, cocktails of new ARVs proved so effective that an HIV infection became a chronic disease. Brown changed regimens repeatedly to take advantage of the advances, but he suffered from many side effects, from headaches and night sweats to diarrhea and vomiting. In 2000, his doctor suggested he take a break and stop all medication.
By 2002, Brown's CD4 level had plummeted to 250, 50 shy of an AIDS diagnosis, which indicates that a person's immune system can no longer contain otherwise harmless opportunistic infections. He started treatment again, easily tolerating a cocktail of new-generation ARVs. The amount of virus in his blood, the so-called viral load, dropped to an undetectable level on the most sensitive test available.
Four years later, Brown, then 40, received a second devastating diagnosis: He had developed acute myeloid leukemia, a highly lethal cancer, unrelated to HIV. Because of the complexity of treating leukemia in an HIV-infected patient, his doctor referred him to Campus Benjamin Franklin, a large clinic affiliated with the Free University of Berlin. Gero Hütter, an oncologist and hematologist, took the case.
Three rounds of chemotherapy caused liver and kidney failure and high fevers; Brown became so ill that the hospital put him in the intensive care unit and induced a coma. “While I was in the coma for 16 hours, they thought I was going to die and told my boyfriend they didn't know if I would make it,” Brown says. But he fully recovered. In September 2006, Brown took a monthlong trip to Italy and upon his return started working out at the gym again. “I was doing fine,” he recalls.
Hütter, who has since moved to the University of Heidelberg, warned his patient that leukemia often returns, and that if it did, the next line of treatment was a bone marrow transplant to replace his stem cells. Then Hütter, who had never treated an HIV-infected patient before, proposed a radical idea: What if a stem cell transplant could eliminate both the leukemia and HIV?
Hütter knew from reading the literature that some rare individuals were highly resistant to becoming infected with HIV because of a mutation in their CD4 cells. HIV establishes an infection by attaching to both the CD4 receptor and a second one on the cell known as CCR5. A mutation in the CCR5 gene known as δ32, which causes no obvious harm, bars the cellular doors to HIV. Hütter suggested to Brown that if they had to do a transplant, they might as well try to find a δ32 donor. “I told him we don't know what will happen, but there might be a chance we'll get rid of the HIV,” Hütter says.
Brown at first wasn't interested. “I thought I had got rid of the leukemia because the chemotherapy had worked,” Brown says. “And I figured that I always had the medication to fall back on for HIV, and I could probably take it for 50 years and do OK.” But in early 2007, his leukemia relapsed, and he gave Hütter the green light to look for a δ32 donor. After screening 62 possibilities for a genetic match that Brown's immune system would tolerate, Hütter found a German man living in the United States who had inherited the δ32 mutation from both parents. They transplanted Brown in February 2007, a procedure that required first destroying his immune system with drugs. He stopped taking his ARVs.
Eleven months later, his leukemia returned again, but his HIV remained undetectable. Brown received a second transplant from the same donor, this time first ablating his immune system with drugs and whole-body irradiation.
Today, Brown remains free of leukemia. And although he has not taken ARVs for more than 4 years, the most sophisticated labs in the world cannot find any HIV in his body
At a press conference in November 2008, Hütter described his patient's promising prognosis.
CREDIT: JOHANNES EISELE/REUTERS
Proof of concept
The AIDS epidemic surfaced in June 1981, and the virus has since infected more than 60 million people, killing half of them. Brown, who became widely known in the media as the “Berlin patient” after Hütter publicly described the case in 2008, is the only living human, a growing consensus contends, to be cured.
Since 2008, there's been widespread leeriness about oversimplifying Brown's case and hyping the prospect of an AIDS cure. And Brown's treatment clearly does not offer a road map for many others. After all, the expensive, complex, and risky transplant only made sense because Brown was dying from leukemia. Nor is it clear exactly which components of the extensive transplant regimen cleared the virus from his body.
But Brown's case has moved the much-ridiculed cure idea onto the most scientifically solid ground it has yet occupied, say leading AIDS researchers. Brown's case showed for the first time that it is possible to rid the body of the virus—even from the minuscule reservoirs where the virus can hide out for years, evading both the immune system and ARVs. His astonishing turnaround also raised hopes that other, more practical drugs and immune system modulators might find and destroy every last bit of virus—or at least reduce it to such low levels that people no longer need ARVs.
There are skeptics who say Brown's virus may still come back. At first, even Hütter would not say Brown had been cured, pointing out that a cancer patient has to be free of disease for 5 years before an oncologist will use that term. But he is increasingly willing to use the “c” word, as are other leading researchers.
“Some people pooh-pooh it, but I think it's a game changer,” says James Hoxie, a virologist at the University of Pennsylvania. “It's put the word ‘cure’ in the vocabulary. I don't care if it's n = 1. It shows that it's possible.” Steven Deeks, an HIV/AIDS clinician at the University of California (UC), San Francisco, who has put efforts to cure the disease on the front burner, says, “A lot of people were interested in cure research before the Berlin patient, but he is a proof of concept. That had a big impact on the field.”
Millions of new dollars are being devoted to what some call “a race” to find a cure. The U.S. National Institutes of Health (NIH) last November solicited proposals for an $8.5-million-a-year collaborative grant to search for a cure, and several high-powered consortia applied. NIH last month added another $4.5 million to the cure research pot with the announcement of new grants to develop therapies that will allow people to stop taking ARVs for prolonged periods. The Bill and Melinda Gates Foundation, the California Institute for Regenerative Medicine, and the Foundation for AIDS Research has each issued cure-related grants. The push for a cure is further intensified by limitations of even the best ARVs and the extraordinary cost of providing them to everyone in need for decades on end.
Christine Katlama, who heads the AIDS clinical research unit at the Pitié-Salpêtrière Hospital in Paris, said she hasn't felt this buoyed about the prospects of helping HIV-infected people since the arrival of powerful ARVs—“highly active antiretroviral treatment,” or HAART—15 years ago. “For many of us who were for a long time in the field of HIV, it's a very exciting time,” she told the 200 attendees at the International AIDS Society's first-ever meeting devoted to cure research, which was held in Vienna last July.
Purging reservoirs
In 1996, Time named AIDS researcher David Ho its “Man of the Year,” in no small part because he had dared to proclaim that HAART might eliminate HIV from a person's body. Ho, head of the Aaron Diamond AIDS Research Center in New York City, calculated that given the half-life of immune cells, if HAART completely suppressed the virus for 3.2 years, eradication would likely occur. But in the spring of 1997, Robert Siliciano's group at Johns Hopkins University in Baltimore, Maryland, reported a pivotal discovery that torpedoed Ho's simple cure idea. It also introduced a formidable challenge to curing the disease that continues to this day.
Siliciano's group identified the existence of pools of “resting” CD4 cells that harbor HIV in their chromosomes but do not produce new viruses unless they are called into action. These latently infected cells serve as the immune system's memory and can live for years. Siliciano and Ho together published a study in the 14 November 1997 issue of Science (p. 1295) showing that people on HAART who had “undetectable” levels of HIV on standard tests for up to 30 months all had reservoirs of about 1 million latently infected cells. Siliciano, his wife Janet, who is also at Johns Hopkins, and co-workers later estimated that it would require 72 years of HAART to clear those stubborn reservoirs.
As bleak as his assessment was, Siliciano's discovery pointed the way to a new strategy for curing AIDS. He and other investigators reasoned that if they could wake up those resting cells, they would start to divide and force HIV out of hiding. This, theoretically, would “purge” the reservoir as the production of HIV would either kill the cells directly or mark them for immune attack. ARVs would mop up any new HIVs before they could infect virgin cells.
A few groups, including Ho's, soon tried to purge reservoirs by jolting the immune system with agents like the chemical messenger interleukin-2 and a monoclonal antibody. Virologist Douglas Richman of UC San Diego says this strategy caused the equivalent of toxic shock syndrome. “They almost killed their patients,” Richman says.
They almost killed cure research, too. But with Brown's success and a more refined understanding of reservoirs, efforts to purge them are again the cornerstone of cure strategies. Several prominent research groups have begun experimenting with a variety of different approaches, including using drugs already on the market to treat other diseases, tweaking immune systems, and engineering gene therapies to mimic Brown's transplant.
All these early trials are big gambles, and no one is expecting to discover a cure anytime soon. But with the field's momentum and better funding, hopes are high that progress—which at this point would mean simply showing that an intervention reduced a latent reservoir by any amount—could surface in the next year or two. And luckily for the field, there are plenty of HIV-infected people willing to join these cure studies, which often means agreeing to highly unusual experimental interventions and the extremely invasive tests needed to assess whether they have had an impact.
“It's not like you can take a little blood from someone's arm and know something. … To measure a rare event you need a lot of cells.”
—DAVID MARGOLIS, UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL
CREDIT: J. COHEN
Acute angles
At 8 a.m. one October morning in 2010, a researcher dressed in a coat and tie entered the apheresis room at the memorial hospital run by the University of North Carolina (UNC), Chapel Hill. But the man, who is in his mid-50s, had not come to study the patients in this room—he was one of them. Over the next 4 hours, a machine would drain the blood from his body two times, 10 liters in all, and replace it, separating out billions of his white blood cells for later analysis. The researcher, who asked that his name not be used, had joined a unique study at the university led by UNC molecular virologist David Margolis that hopes to shrink the HIV reservoir.
Four years earlier, the researcher had gone to see his doctor after repeated night sweats and a fever spiking to 40°C. “I told him I'd been involved in risky behavior and should be tested for HIV,” recalls the man, who is married and has children. The antibody test came back negative.
The researcher knew that an HIV infection could take several weeks to trigger a detectable antibody response used in the stock test, but that the polymerase chain reaction could pluck out the viral genetic material itself. He requested a PCR test, which confirmed that an acute HIV infection explained his flulike symptoms. “The first thing I thought about was suicide, the second thing I thought about was suicide, and the third thing I thought about was suicide,” he says.
Within a month, the man joined a trial at UNC to assess whether HAART offered extra benefits if people started treatment during acute infection. In particular, HIV destroys CD4s in the gut during the first few weeks of infection, and evidence suggests that when people wait to start HAART—which is standard care—those cells never completely return. This in turn causes a chronic state of inflammation that leads to long-term health problems such as cardiovascular disease and diabetes, even in people who have undetectable levels of virus while on HAART.
The UNC team is now following 80 people who started ARVs during acute infection, and from that cohort, they have recruited 20 volunteers, including this man, to take part in a cure trial that began in March. Margolis and his team reasoned that people treated during the acute phase should have smaller pools of latently infected cells than those who start HAART later, and the smaller the reservoir, the easier it should be to drain.
The UNC study adds to standard HAART a Merck drug, SAHA, already on the market to treat cutaneous T-cell lymphoma. In addition to its cancer-fighting abilities, SAHA tinkers with the structure of chromosomes and kick-starts transcription of DNA (see sidebar), which Margolis and others are hoping will flush latent HIV out of hiding, leading those cells down the road to ruin.
Determining whether SAHA works requires an extraordinary commitment from the infected researcher and the 19 other participants: These otherwise healthy people must repeatedly undergo the grueling, time-consuming apheresis procedure to quantify their minuscule reservoirs and assess whether they have shrunk. “It's a big hassle,” Margolis says. “It's not like you can take a little blood from someone's arm and know something. … We're trying to measure a rare event, and to measure a rare event you need a lot of cells.”
Daria Hazuda, vice president of virus and cell biology at Merck Research Laboratories in West Point, Pennsylvania, shares Margolis's enthusiasm that SAHA might shrink reservoirs. But Hazuda, who is part of a collaboration led by Margolis that's bidding for the new NIH cure money, also urges people to keep hope in check. “From an experimental medicine point of view, that's a great approach,” Hazuda says. “Is that going to be the answer by itself? Having worked so many years on HIV therapy, it would seem to me a single molecule to address reservoirs is a bit naïve.”
Multipronged attack
Don Howard, a 47-year-old management consultant in San Francisco, started on HAART the day after he tested positive in 1996. “At some level, I'm a control freak,” says Howard, who is so fit and radiant he could be in advertisements for energy drinks. “The notion that I could take something to make it all better was extremely appealing.” Now he's first in line for one of the cure studies getting under way at San Francisco General Hospital, where he has been Deeks's patient for 15 years.
Like the UNC group, Deeks and his collaborators hope to purge reservoirs with drugs that trigger transcription, but they have several other strategies in the works that hit the virus from other angles. The trial participants again will come from a group of patients who started treatment during acute infection, and no one has been on HAART longer than Howard. “It suggests he's closer to eradication than anyone in our cohort,” Deeks says.
Howard's motivation to join the study has more to do with curiosity and a desire to contribute than concern about long-term damage from the virus or drugs. “It's more intellectually interesting than it is practically compelling,” he says. Nonetheless, he, too, is willing to undergo deep probes of his body to quantify his reservoir. One of the most difficult challenges researchers face is that reservoirs can pool in tissues that are difficult to access. Howard has already had lymph nodes excised and bone marrow extracted, and he may undergo biopsies of his gut, which is home to an estimated 80% of the body's CD4 cells.
Deeks and his collaborators contend that reservoirs persist by different mechanisms, which will require different strategies to deplete them. One possibility is that even in people like Howard who have undetectable virus on HAART, HIV constantly replicates at low levels, creating new latently infected cells and perpetually refilling reservoirs. Adding another powerful ARV to the HAART cocktail, then, might shut down that production.
“You can't cure people by getting rid of low-level replication, but if you want to cure people, you have to get rid of it.”
—STEVEN DEEKS, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
CREDIT: BILL BERRY
Although several “intensification” studies have failed to detect any impact on HIV, Deeks remains convinced that the approach shows promise. “You can't cure people by getting rid of low-level replication, but if you want to cure people, you have to get rid of it,” Deeks says. He also points to an intensification study reported in the April 2010 issue of Nature Medicine that found hints of an effect. Led by Maria Buzón the Autonomous University of Barcelona in Spain, the study has sparked debate and divided Deeks and his like-minded colleagues from Margolis, Siliciano, Richman, and many others who are convinced that standard HAART has reached the limits of what ARVs can do.
After upping the subjects' ARVs, Deeks's team plans to begin testing the drug disulfiram, also known as Antabuse, which is on the market to treat alcoholism. Siliciano's lab recently discovered in test-tube experiments that disulfiram, like SAHA that Margolis is testing, turns on HIV transcription in latently infected cells, although the mechanism is not clear.
Triggering transcription with drugs like disulfiram or SAHA carries risks, especially when tested in healthy people like Howard. On top of the direct toxicities of the drugs, Eric Verdin, a virologist at the Gladstone Institute of Virology and Immunology and one of Deeks's collaborators, cautions that inducing transcription could stir endogenous retroviruses from their slumber. These remnants of ancient infections constitute 8% of the human genome, and no one knows what havoc they might wreak. “A lot of mechanisms used to suppress endogenous retroviruses are the same mechanisms that suppress HIV,” Verdin says.
In a completely different tack, Deeks's group wants to tweak the immune system to help cure people. One strategy aims to purge reservoirs by hijacking an immune actor with the macabre name programmed death 1 (PD-1). This receptor on the surface of CD4 cells helps slam the brakes on cell division, maintaining the latent state. A test-tube study presented at the 2011 Conference on Retroviruses and Opportunistic Infections (CROI) by two of Deeks's collaborators, virologist Nicolas Chomont and immunologist Rafick-Pierre Sékaly of the Vaccine & Gene Therapy Institute of Florida in Port St. Lucie, showed that when they blocked the PD-1 receptor with an antibody, the cells coughed up gobs of HIV.
Another approach assumes that purge strategies will not eradicate HIV but that shrinking reservoirs in combination with fortifying the immune system could lead to a “functional” cure that allows people to stop ARVs. “There might be a threshold level where the immune system is able to contain the virus,” Verdin says. To that end, the group has joined the multisite Eramune trial (see table, p. 789), which pairs intensification with an HIV vaccine made by NIH.
Designer genes
In a clinical sense, Matt Sharp differs in several ways from Howard and the HIV-infected researcher in North Carolina. “I've been through the ringer and knocked on death's door,” says Sharp, 54, an HIV/AIDS educator who lives in Brisbane, near San Francisco. His face is sunken from lipodystrophy, a side effect of the first-generation anti-HIV drugs. For many years, HIV outwitted his drugs and the virus battered his immune system. He has had two hospital stays because of severe pneumonia and has also battled bouts of tuberculosis (TB) and wasting disease. But he, too, is taking part in pathbreaking cure studies.
Sharp, who works with the San Francisco–based advocacy group Project Inform, learned of his HIV infection in October 1988 when he was a ballet dancer in Oklahoma City. “Who knows when I was infected,” he says. “We all were having unsafe sex because we didn't know any better. Those were back in the days you'd get a sexually transmitted disease and get a shot and you were back into the mix of things the next few days. There wasn't any consideration of death, so we didn't take it that seriously.” His initial CD4 count: 409. “It scared the hell out of me,” he says.
Despite starting treatment a year after he tested positive, in 1991 Sharp developed extrapulmonary TB, a dangerous form of the disease linked to HIV. He moved to San Francisco and plugged in with activist groups that kept him abreast of treatment advances, and as new ARVs became available, he changed his regimen repeatedly. Yet by the mid-1990s, his 6-foot-tall frame had withered from 185 pounds to 155. In 1996, Sharp's CD4 cells dropped to as low as 15. After quickly developing resistance to the protease inhibitors that then formed the cornerstone of HAART, he joined a highly unconventional trial and received a thymus transplant, which putatively would rebuild his immune system. “It wasn't anything that probably helped,” he says.
In 2001, Sharp became one of a small group of people to take injections of Fuzeon, the first drug to come to market that jams the HIV entry process. “It was very painful, but I was willing to do it because I had no options,” he says. His viral load became undetectable for the first time. His good fortune lasted all of 1 week. Five years ago, he added raltegravir to his regimen, and his HIV has subsequently remained undetectable with a CD4 count hovering around 300. “It isn't great, but it got me out of the danger zone,” he says.
Last June, Sharp joined one of two gene therapy studies now under way that tries to mimic Brown's success in relatively healthy HIV-infected people. The small trial, led by Jacob Lalezari of Quest Clinical Research in San Francisco and Ronald Mitsuyasu at UC Los Angeles, sidesteps the need to find an immunologically matched donor who has the CCR5 mutant and does not use ablation. Sharp, known as patient 01-203, and eight other participants first went through leukapheresis to remove about 10 million of their CD4 cells, which the researchers then modified with adenoviruses engineered to contain what are known as zinc-finger nucleases. Developed by Sangamo BioScience in Richmond, California, the zinc-finger nucleases clip out a specific small sequence of DNA in the CCR5 gene, rendering cells incapable of producing a functioning version of the receptor.
After expanding the cells in culture, the researchers in September infused Sharp with about 2 billion of his own rejiggered CD4s. Sharp sat in the audience at the 2011 CROI when Lalezari revealed early results from patient 01-203 and five others. “There are a lot of unanswered questions, obviously, but it was incredibly exciting to see my data points,” Sharp says.
SOURCE: COMPILED BY J. COHEN
As Lalezari explained, the gene therapy modified only about 25% of the cells that they infused, but the CCR5 mutants persisted in Sharp and four of the others for more than 3 months. Rectal biopsies showed that the modified cells had trafficked to the gut, indicating that they had spread to this critical viral hangout. CD4 counts jumped an average of 100 cells. There were no serious, lasting side effects.
This does not come close to a cure. As Lalezari noted, the fraction of modified CD4s eventually dropped to just over 5%, in contrast to Brown, who had 100% of his CD4 cells destroyed and replaced with CCR5 mutants. Lalezari cautiously concluded that the therapy ultimately “offers the hope of providing a protected reservoir of CD4+ T cells that are resistant to HIV infection.”
In a best case scenario, the modified cells will copy themselves indefinitely and evolve into the predominant group. If HIV does replicate in people like Sharp after they have received the gene therapy, the virus should selectively infect and kill the normal CCR5+ cells, and the population of CCR5− mutants would steadily rise. A report in the August 2010 issue of Nature Biotechnology showed that just such a selection occurred in mice engineered to contain a humanlike immune system. Led by Paula Cannon of the University of Southern California in Los Angeles, the experiment used the same Sangamo zinc fingers to cripple CCR5. The transplanted mice then received injections of HIV, and 12 weeks later, the CCR5− mutants almost completely replaced the CCR5+ cells. “And guess what: The virus goes away,” says Cannon, who in October 2009 won a $14.5 million grant from the California Institute for Regenerative Medicine to take a similar approach into the clinic with collaborators at the City of Hope in Duarte, California.
Ultimately, transfused patients will have to stop taking ARVs to see whether their modified immune systems are strong enough to contain the virus. “The great news is we can always put people back on ARVs if the gene therapy fails,” Cannon says. “It's a no harm, no foul strategy.”
In a small trial, similar to the Quest study, that's run by Carl June of the University of Pennsylvania, two patients who received the zinc-finger gene therapy went off ARVs, with intriguing results. As June reported at the 2011 CROI, HIV returned in both patients, but in one, the virus took 10 weeks to surface, a signal that the modified cells made life more difficult for HIV.
Sharp has no interest in going off his medication. The infusion has boosted his CD4s from the dangerously low 281 to the 600 range—the highest they have been since he was diagnosed 23 years ago. He's quick to point out that it's unclear whether the CD4s are functional and improving his health, but he is convinced that, come what may, his contribution will help advance this “very sexy area of research.” “Those of us who have survived this long with HIV are able to look back and say, ‘Why shouldn't there be a cure?’” Sharp says. “I'm so passionate about this because I want to see a cure before I die.”
Beginning of the end
If researchers do eventually find a widely applicable cure for HIV, the process likely will resemble Brown's rough-and-tumble life, which has seen a remarkable success set against a backdrop of dramatic setbacks. “We are really at the very rudimentary stages,” says UC San Diego's Douglas Richman. “If there is going to be success—and I'm optimistic—it's going to be 1 to 2 decades.”
The field will receive a big boost in July with NIH injecting millions of new dollars to up to two collaboratory groups working on cure research. There's also a high-profile campaign led by the International AIDS Society and its president-elect, Françoise Barré-Sinoussi—who won the Nobel Prize for co-discovering HIV—to organize the research agenda and then seek more funding. “We are in a period of economic crisis, but if we work together, we can do a lot,” Barré-Sinoussi says. On the advocacy front, the Philadelphia-based AIDS Policy Project is aggressively pushing the agenda, lobbying the U.S. Congress and NIH to make cure research a top priority.
Brown, who recently moved to San Francisco, says he initially did not imagine his transplant of CCR5 mutant cells would have much impact on others. “At first, I was like, nobody can get this, it's not very practical, and I don't think anybody could use it for curing HIV.” But he now sees his singular triumph as having played a catalytic role in advancing the once-laughable idea that research could discover a cure for this intractable infection, and he's grateful for the chance to give back. “I'd like to be able to continue to tell people about my situation,” says Brown, who hopes to write a memoir. “It's a feeling that I'm doing some good for the world.”
（原载科学杂志：http://www.sciencemag.org/content/332/6031/784.full）
