# News this Week

Science  14 Sep 2007:
Vol. 317, Issue 5844, pp. 1480
1. SPACE PHYSICS

1. Andrew Lawler

Dark energy is a subtle force believed to be responsible for accelerating the universe's expansion. Last week, it also proved irresistible to a panel of U.S. physicists and astronomers asked to set priorities for an ambitious set of modestly priced missions to tackle the most exciting cosmological questions of the era. Its report recommends that NASA and the Department of Energy (DOE) begin work next year on a $1-billion-plus Joint Dark Energy Mission (JDEM), while saying that plans for four other space physics spacecraft should be delayed—some indefinitely, given the pressure on NASA's science budget. Four years ago, cosmologists came up with five distinct projects to examine black holes, gravitational waves, dark matter, the early inflation of the universe, and dark energy as part of what NASA labeled its Beyond Einstein program. Faced with tight budgets and prompted by congressional concerns, the agency last year asked the National Academies' National Research Council (NRC) for advice on deciding which missions should get off the ground first, starting in 2009. The council's 220-page report, issued 6 September, gives top billing to the dark energy effort, followed by the Laser Interferometer Space Antenna (LISA), a three-satellite mission designed to detect gravitational waves. The rest of the projects, says the report, will have to wait their turn. Making a queue for these missions is critical given the limited funding for new science initiatives at NASA. The NRC panel, led by Charles Kennel, an atmospheric scientist and physicist at the University of California, San Diego, also broke new ground by developing independent cost estimates for missions that are not yet in NASA's pipeline—and by concluding that each project team had seriously underestimated the cost of building and operating the spacecraft. It said Constellation-X, an advanced x-ray telescope, for example, would cost$3 billion rather than $2.1 billion. And with a program budget expected to rise from only$37 million in 2009 to $211 million in 2012, Beyond Einstein seems incapable of supporting more than one mission in the near future. “Our task was to address a mission which could fit [into a] budget wedge opening up in 2009,” Kennel explained to reporters. JDEM's success was due to both its scientific appeal and the maturity of its technology, panel members said. DOE's promise of up to$400 million didn't hurt. Saul Perlmutter, a DOE Lawrence Berkeley National Laboratory physicist and co-principal investigator on one of the three different proposed versions of the project, says that JDEM could be launched by 2015 and that the project has benefited from $40 million from DOE during the past 3 years. LISA, estimated to cost$2.75 billion rather than $2 billion, is another project counting on outside funding, with the European Space Agency (ESA) offering to foot$600 million of the bill. Kennel says the panel found LISA “an enchanting and technologically exciting mission” but suggested that NASA make no further plans until after the Pathfinder spacecraft, an ESA/NASA mission that will test LISA technologies, flies in late 2009. “We felt that in the long term, LISA will be the Beyond Einstein flagship,” Kennel noted. “But it will not be ready in 2009.”

In contrast, the NRC panel recommended kicking Constellation-X out of the Beyond Einstein tent because its contributions to science are likely to extend beyond the scope of the initiative. “Beyond Einstein is not the sole justification [for Constellation-X] or its primary benefit to the science community,” the report concludes. Another mark against it is a price tag that is comparable to one of NASA's major observatories, and $900 million above the previous projection. Bringing up the rear are the Black Hole Finder Probe and Inflation Probe, a ranking that didn't surprise backers of those two projects. “The competition was intense,” says Harvard University astrophysicist Jonathan Grindlay, principal investigator on one of the two black-hole projects. The committee found a number of problems with the probe, which is designed to find black holes of all sizes, including the difficulty in pinpointing low-luminosity black holes, questions about whether it could accurately determine growth rate of black holes, and uncertainty in identifying the galaxies in which they reside. It also gave a cost that's roughly double the initial$1 billion estimate. Grindlay disputes the new price tag, which he calls “way out of line,” and adds that the committee ignored recent findings on high-redshift gamma-ray bursts that are beacons for black-hole creation.

Other researchers give the NRC panel high marks for weighing the science that could be done before considering schedules and cost. “There were so many good ideas, they had a tough choice,” says Bradley Schaefer, an astrophysicist at Louisiana State University in Baton Rouge. NASA officials were also pleased with the results. “We're happy with what they've accomplished,” says Jon Morse, NASA astrophysics chief, emphasizing the importance of a fiscally realistic plan. “We need to contain irrational exuberance.”

2. U.S. NATIONAL SECURITY

1. Yudhijit Bhattacharjee

For more than 3 decades, U.S. science agencies have used images taken by the nation's spy satellites to study everything from erupting volcanoes to the migration of marine mammals. Now, a new plan to expand the use of the satellites for homeland security and law enforcement has left some officials worried that science will suffer.

Last month's announcement by the Department of Homeland Security (DHS) that it was setting up a new National Applications Office (NAO) this fall to widen the use of spy-satellite imagery has sparked protests from civil liberties advocates. They worry that federal, state, and local authorities will seek high-resolution, real-time images to monitor activities of U.S. citizens in the same way that the satellites help track terrorist activities overseas. But officials at federal science agencies are concerned for a different reason: They suspect that the new arrangement could mean fewer chances to investigate scientific questions or cause delays that undermine the value of the information.

The satellites are operated by defense agencies and used mainly for reconnaissance overseas. Federal scientists can ask for permission to see specific images—as well as request that specific images to be taken—by applying to the Civil Applications Committee (CAC). Recommendations from the committee, which is headed by the director of the U.S. Geological Survey (USGS) and includes officials from more than a dozen agencies, are reviewed by the National Geospatial-Intelligence Agency (NGA), which oversees military and intelligence mapping efforts. Last year, CAC forwarded about 50 such requests.

Researchers have used the program to access images of phenomena such as the movement of glaciers in Yakutat Bay in Alaska, forest fires in Montana, and Mount Pinatubo in the Philippines. “If we are concerned that a volcano is about to erupt, we would like to be able to get the data now,” says James Devine, an adviser to USGS director and CAC chair Mark Myers.

Under the new plan, CAC will report to NAO in parallel with two new working groups that will serve homeland security and law enforcement. NAO will take requests from all three working groups and pass them on to NGA, essentially adding a layer of bureaucracy. Some CAC members fear that scientific requests will end up at the bottom of the queue, far behind requests such as aerial images of vehicles at the U.S.-Mexico border. Science officials are also concerned that disagreements over privacy could lead Congress to decide that only intelligence agencies can use the data.

Some legislators have expressed similar apprehensions. In a 16 August letter to DHS Secretary Michael Chertoff, Representative Edward Markey (D-MA) asked the agency to describe how it plans “to ensure that vital scientific activities are not eroded” as the program expands “to include homeland security objectives.”

DHS officials say those fears are unfounded. Speaking to Science at a House hearing last week on the new office, Charles Allen, DHS chief intelligence officer, said that the scientific program “is going to become more robust than ever. We are going to work hard for all of our customers, including science agencies.”

CAC members say they won't know if science is being served until they learn more about the new office. “The people in charge have been a group of scientists representing their respective agencies,” says another CAC member who requested anonymity. “With the program moving toward some sort of a domestic surveillance mission, can the science continue as before? I don't know.”

3. GENOMICS

# A Little Gene Xeroxing Goes a Long Way

1. Jon Cohen

Researchers studying the evolution of starch digestion have uncovered evidence of a surprising adaptation: Rather than relying on mutations in a particular gene to help us digest roots and tubers better, the human genome simply made more copies of the gene in question. The finding is one of the strongest examples yet of evolution affecting gene copy number in humans and sheds light on how our diet split us apart from other primates.

An enzyme called salivary amylase—encoded by the AMY1 gene—helps humans digest starchy food. In a typical evolutionary scenario, natural selection would favor random mutations in AMY1 that caused it to churn out more of the enzyme or a more effective version of it in people who ate a high-starch diet.

But a study published online 9 September in Nature Genetics contends that something else happened. Nathaniel Dominy, an evolutionary anthropologist at the University of California, Santa Cruz, and George Perry at Arizona State University in Tempe analyzed AMY1 in high-starch eaters such as Americans of European descent, Japanese, and Hadza from Tanzania, hunter-gatherers who eat many roots and tubers, as well as groups that eat little starch, such as the Biaka of the Central African Republic and the Mbuti from Congo, both rainforest hunter-gatherers, and Tanzania's Datog and Siberia's Yakut pastoralists. In all, the researchers studied samples from more than 200 people.

The team found that rather than having mutations that boosted AMY1's activity, the high-starch eaters had extra copies of the gene. On average, the high-starch eaters had seven copies of the gene, whereas the low-starch populations had only five. “If you have a gene that's working well, why not just copy it over and over again?” asks Dominy. “Why wait for evolution to just roll the dice?”

For a broader evolutionary perspective, the researchers looked at 15 chimpanzees, which eat little starch. All had only two copies of AMY1. And an analysis of the gene from bonobos, the chimp's closest relative, found that it had mutations that may prevent AMY1 from functioning altogether. “I was very excited to see this,” says Gregory Laden, a biological anthropologist at the University of Minnesota, Twin Cities, who contends that eating starch-rich roots and tubers played a key role in differentiating humans from other apes.

Ajit Varki, who studies human origins at the University of California, San Diego, says the report also suggests that humans may have had access to starchy foods before the advent of agriculture, as is commonly thought. Even populations with low-starch diets had extra AMY1 copies, he notes: “This would imply that first there were some rounds of duplication of the gene in preagricultural humans, and then that went further in agricultural humans.”

4. CONSERVATION

# Scientists Say Ebola Has Pushed Western Gorillas to the Brink

1. Gretchen Vogel

The combined threat of the Ebola virus and poaching have pushed western gorillas into the “critically endangered” category in the latest international ranking of species threatened with extinction. Although estimates suggest that tens of thousands of the animals still live in west-central Africa, the new Red List from the World Conservation Union (IUCN) moves the species into its highest alert category, in large part because of fears that continuing Ebola outbreaks could swiftly wipe out still-significant gorilla populations.

The list, released on 12 September, highlights the western gorilla as well as dozens of other species for which new data indicate an increased risk of extinction. The “critically endangered” category is usually applied when just a few hundred individuals survive in the wild. But researchers say that western gorillas, despite their relatively large numbers, are in serious trouble. An ongoing series of Ebola outbreaks has killed up to 90% of the animals in some regions (Science, 8 December 2006, p. 1522), and the use of vaccines to stem the disease faces daunting challenges. Adding to the pressure, the rapid development of logging roads has opened up vast new regions to poaching and the bush-meat trade.

Although the other species in the Gorilla genus, the eastern gorilla, is far less numerous than the western gorilla, IUCN ranks the former one level lower at “endangered” because it is outside the current area of Ebola outbreaks. As for western gorillas, there may be as many as 30,000 left in their current range, which stretches across Gabon, Equatorial Guinea, and parts of Cameroon, the Central African Republic, and the Republic of the Congo. There are two subspecies, the more common western lowland gorilla and the extremely rare Cross River gorilla, of which fewer than 200 probably remain.

It is unusual for disease to be cited as a reason for reclassification, says wildlife disease specialist Richard Kock of the Zoological Society of London, who co-chairs the IUCN Veterinary Specialist Group. But even if the new status has come sooner than expected, the change is warranted, says Kenneth Cameron, a field veterinarian with the Wildlife Conservation Society in Brazzaville, Republic of the Congo. “Is this jumping the gun a bit? Some would argue that it is,” he says. “But it is inevitable that this species is going to end up on a critically endangered list. It's simply a matter of when.”

IUCN experts found that the western gorilla population has declined by 60% in the past 20 to 25 years and estimated that in the past 15 years Ebola has killed one-third of the animals living in protected areas such as national parks. Those numbers are only the roughest of estimates, Kock says. The current gorilla range “is a huge place. … It's bloody impossible to know what's going on” in the remote forest regions, he says.

Conservationists say they hope the new status will help pressure governments and international donors to increase efforts to protect gorillas and their habitat. They also say they hope it will lead to more funding for the search for an Ebola vaccine.

What is certain is that western gorilla habitat will be under severe pressure in the next 5 years, Cameron says. Plans are under way in the Republic of the Congo to improve the road and rail connections between Brazzaville and Ouesso, the largest town in the north. Both projects will cut through prime gorilla habitat, making it easier for hunters to reach and for bush meat to be shipped back to city markets.

While public awareness campaigns and increased antipoaching efforts might help mitigate pressure from hunters, scientists are struggling to blunt the impact of Ebola. The virus can pass from ape to ape, so regions with higher population densities are especially at risk. “It appears to act like a brushfire,” Cameron says. “You get a lightning strike somewhere, and it starts to burn.”

Although admitting it's a long shot, some researchers hope a vaccine campaign could at least save enough animals to preserve the species. At least half a dozen vaccine candidates have protected mice or monkeys in the lab from the Ebola virus. But finding a way to deliver a vaccine safely to wild animals is no small challenge.

Few believe that vaccine-laden darts could reach enough gorillas to stem the spread of the disease. Peter Walsh of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, is working with a vaccine company to develop possible baits that could carry an oral vaccine. The bait must keep the vaccine viable in the hot, humid conditions of the forest, attract great apes, and be safe for other animals who might find it first. Walsh says that before the end of the year, he and his colleagues plan to begin testing darting and oral bait strategies, without incorporating a vaccine, in the Republic of the Congo.

“This is not just about Ebola,” Walsh says. “All apes are under increased disease threat, especially from human-introduced diseases. Vaccines are going to be an increasing part of conservation. … This is not going to be wasted time or money.”

5. EPIDEMIOLOGY

# Tropical Disease Follows Mosquitoes to Europe

1. Martin Enserink

For years, medical entomologists have worried that the astonishing ascent of the Asian tiger mosquito (Aedes albopictus) might bring not only nasty bites but also new public health surprises. After all, the mosquito is a known vector for more than 20 viral diseases.

They were right. This summer, the mosquito, which has become firmly established in southern Europe, has infected almost 200 people in Italy with chikungunya, a painful viral disease. It's the first known example of chikungunya transmission outside the tropics—and it's making scientists wonder whether A. albopictus has the potential to touch off much larger outbreaks in Europe and the United States.

Chikungunya is rarely fatal but can cause severe fevers, headaches, fatigue, nausea, and muscle and joint pains. People started falling ill in Castiglione di Cervia and Castiglione di Ravenna—two villages separated by a river in the province of Ravenna—in early July, says Antonio Cassone of the Istituto Superiore di Sanità (ISS), a national government lab in Rome. But most patients' symptoms were mild and resembled those of other diseases, such as the Toscana virus, so health officials didn't notice for a while. Samples reached ISS on 27 August, and the virus was identified the next day.

Epidemiological detective work suggests that the index patient was a man who traveled to one of the villages and became sick there, after having been infected in India. Isolation and sequencing of the virus are under way to confirm that theory, Cassone says. One patient, an 83-year-old man with severe preexisting medical problems, has died.

Chikungunya sickened more than one-third of the almost 800,000 inhabitants of La Réunion, a French island in the Indian Ocean, in 2005 and 2006 (Science, 24 February 2006, p. 1085). India suffered an explosive outbreak in 2006 with more than 1.25 million cases, although some believe the real toll is much higher. Several European countries had seen “imported” cases of chikungunya lately, but local transmission in Europe has never been observed before. “It's fascinating,” says entomologist Paul Reiter of the Pasteur Institute in Paris.

A daytime biter, A. albopictus originated in Southeast Asia and has made impressive strides across the globe in the past 2 decades. It was first found in the United States in secondhand tires imported from Asia in Houston, Texas, in 1985; today, it has spread to more than 20 southern and eastern states. In Europe, the mosquito has appeared in Mediterranean countries from Greece to Spain and as far north as the Netherlands. Its eggs often hitch a ride with plants shipped in water containers, such as the popular Lucky bamboo.

It's too early to tell whether chikungunya now has a permanent foothold in Europe. New cases have slowed to a trickle, says Cassone, in part because the mosquito population is dwindling as temperatures drop. A critical question is whether infected mosquitoes can survive the winter or pass on the virus to their offspring via their eggs, says Reiter. “If they can, we might see a rip-roaring epidemic next year,” he says. Even if they can't, any newly imported case could kick off an outbreak in the future.

There are no drugs or vaccines against chikungunya, but the outbreak at La Réunion triggered renewed interest in an old vaccine candidate developed in the 1980s by a U.S. Army lab in Fort Detrick, Maryland. Scientists at three French government institutions are now working on that vaccine, and new clinical trials might begin before the end of 2008, says epidemiologist Antoine Flahault, who chaired a French task force on chikungunya last year.

6. BIOSECURITY

# Reports Blame Animal Health Lab in Foot-and-Mouth Whodunit

1. Martin Enserink

Neglected, leaky pipes and England's record-setting wet summer likely combined to cause the country's recent outbreak of foot-and-mouth disease (FMD), according to two reports issued last week. The virus responsible probably escaped from a company, Merial, that grew vast amounts of it for vaccine production, the studies say. Yet the reports assign most of the blame for the outbreak to the Institute for Animal Health (IAH), a government lab at the same site in Pirbright that owned the aging network of underground wastewater pipes and was aware that it needed maintenance. IAH breached biosecurity in other ways as well, the reports found.

The findings are a blow to the reputation of IAH, a world-renowned FMD research center, says Andrew Mathieson, an environmental health expert at the University of the West of England in Bristol. But they should also serve as a more general warning. “My worry is: What about the many other research establishments of the same age?” he says.

Rapid government action helped contain the FMD outbreak, first confirmed on 3 August, to just two farms in Surrey (Science, 10 August, p. 732). Still, the National Farmers' Union puts the accident's economic impact at more than $100 million, and some politicians have called for resignations at the Department for Environment, Food and Rural Affairs (Defra), which oversees biosafety at IAH and also funds some 65% of its work. Genomic comparisons of the outbreak virus to strains from Merial and IAH can't pinpoint from which of the two labs the virus escaped, according to the reports, one led by the U.K.'s Health and Safety Executive (HSE), a government agency, and the other by molecular epidemiologist Brian Spratt of Imperial College London. Still, the panels say, it's much more likely that the virus came from Merial, which grew it in two 6000-liter vats shortly before the accident, producing a million times more virus than IAH used in its small-scale experiments. But how did it escape? The reports conclude that air leaks, contamination from solid waste, and foul play by terrorists or disgruntled employees are unlikely. Instead, both focus their suspicions on the site's wastewater system. A two-step chemical strategy is used at Pirbright to prevent FMD from escaping in liquid waste. Both Merial and IAH first treat wastewater at their own buildings with a disinfectant such as citric acid. Then, a complex system of pipes takes the water to a shared effluent treatment plant, managed by IAH, where caustic soda is used to raise the pH to 12 and kill off any remaining virus during a 12-hour holding period. Finally, the liquid is released into the sewer. Although the first treatment step probably killed off almost any leftover virus at IAH, it likely didn't inactivate the larger amounts in Merial's wastewater. The second treatment step would normally take care of that, but the network of pipes, pumps, and manholes leading to it suffered from leaks due to cracks, tree roots, and other problems. The reports hypothesize that live virus seeped into the soil as a result, especially because July's excessive rainfall may have caused the drains to overflow. As it happened, construction crews were digging holes around the leaks at the time, and heavy trucks—without proper IAH oversight—drove through the presumably virus-laden mud. Some of these vehicles later took a road that went very close to the first infected farm. From there, the farmer may have carried the virus to his herd. IAH, a part of the U.K. Biotechnology and Biological Sciences Research Council (BBSRC), owns the antiquated drainage system, the HSE report says. It was also aware of some of the network's problems. In fact, IAH, Defra, BBSRC, and Merial had debated an upgrade since 2003; the problem was money. As to Merial's discharge of virus into its wastewater, HSE says this wasn't a breach of biosecurity, because Defra had approved the procedure used in the first disinfection step. But in a statement, IAH pointed its finger at Merial, suggesting that the company should have taken better care to inactivate any virus. Strangely, the Spratt report says, IAH didn't seem to know that Merial might release active virus into the system; biosafety officers from the lab and the company hardly ever talked. Both panels question the wisdom of chemically inactivating wastewater altogether. Indeed, most modern labs use thermal inactivation—that is, pressure-cooking at 121°C—to destroy any pathogens, says Lee Thompson, a biosafety officer at the University of Texas Medical Branch in Galveston. Still, the second step, using caustic soda, “is very effective against FMD,” Thompson says—but underground pipes that cannot be inspected “are a big problem.” Defra says it will adopt a range of recommendations to fix problems at Pirbright, such as keeping better track of visitors and making sure biosafety officers communicate. Merial has agreed not to grow live virus until U.K. authorities give it the green light. IAH, which was constructed in 1924, is due to be almost completely rebuilt by 2012, although some funding issues remain. Defra has also asked Health and Safety Commission chair Bill Callaghan to review the regulatory framework for animal pathogens. He is due to report by December. 7. BIODEFENSE RESEARCH # Lapses in Biosafety Spark Concern 1. Jennifer Couzin An apparent breakdown in biosafety at Texas A&M University (TAMU) in College Station is prompting scrutiny of the expansive U.S. biodefense research program and the assurance that federal inspections keep researchers following the rules. Last week, the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, described a dozen safety lapses at TAMU, from unreported exposure to pathogens to inadequate protective gear. TAMU's research on select agents, pathogens that are considered potential bioweapons, is on hold until it complies with the regulations. But in a twist, CDC may have indicted itself along with TAMU. Toward the end of a 21-page report, it noted that it had inspected TAMU as recently as February and found nothing other than minor problems, such as variation in how staff tracked lab inventory. After prodding by an independent whistle-blower, however, CDC inspectors returned to TAMU in July and uncovered the violations described in last week's report, many from before the February inspection. Biosafety and bioweapons experts say the charges are among the most damning that they can recall. They include three missing vials of Brucella bacteria; unauthorized employees working with select agents; a faculty member performing a recombinant DNA experiment without the necessary CDC approval; concerns about disposal of animals used in select-agent experiments; and three unreported cases of individuals exposed to the bacterium Coxiella burnetii, which causes Q fever, a treatable but infectious disease. “There was no evidence that a coordinated response or biosafety assessment was performed as a result” of these exposures, notes the CDC report. The report “reflects about as badly on CDC as it does on Texas A&M,” says Edward Hammond, director of the Sunshine Project in Austin, Texas, a bioweapons watchdog group. Last spring, Hammond's repeated demand for TAMU documents under a state open-records law revealed that in April 2006, an employee was diagnosed with brucellosis, an animal disease. TAMU did not report the infection to CDC until after it came to light (Science, 20 April, p. 353). Inspectors initially missed the “train wreck of a select-agent program,” says Hammond. CDC has “got some explaining to do.” The agency declined to comment. “There's nothing I can add,” said CDC spokesperson Von Roebuck, when asked whether the agency would speak to its failure to detect the major safety violations in its February inspection of TAMU. One possible explanation, says Ronald Atlas, a microbiologist at the University of Louisville in Kentucky, is that normally, “the inspections are of the lab facility” and equipment, not an in-depth look at lab procedures. Many of the TAMU violations, however, concern access to pathogens and lab practices. The problems CDC cited are serious but probably not unique, according to scientists both inside and outside TAMU. “If you were to apply an equivalent level of scrutiny at other institutions, I think you would find issues of concern,” says TAMU microbiologist Vernon Tesh, one of four lab leaders singled out for safety lapses in CDC's report. “You always have to have safety in mind,” he added. “Having said that, accidents happen.” In a press conference last week, TAMU's interim president Eddie Davis said that other “institutions under that same level of review would probably have findings that would be reportable to the CDC.” Since the CDC's July inspection, the university's vice president of research and overseer of biosafety compliance, Richard Ewing, has resigned from his position and returned to the mathematics department (Science, 17 August, p. 879). Another biosafety official, Brent Mattox, also left his post. Davis declined to assign responsibility for the lapses or say whether any employees would face disciplinary action. He praised Ewing for having “been very loyal and competent.” CDC has passed its report up the ranks to its parent agency, the Department of Health and Human Services, whose inspector general's office will consider whether to levy fines of up to$500,000 for each of the 12 violations. The Government Accountability Office, Congress's investigative arm, is looking beyond TAMU, examining risks associated with the growing number of high-level biosafety labs. The House Committee on Energy and Commerce plans to hold a hearing on the subject in early October.

Meanwhile, scientists wonder what effect the TAMU findings will have. “Biosafety is mandated by the public, or they're not going to let us do this research,” says Atlas.

“This looks bad for all of us,” says Philip Hauck, a biosafety professional in New York City. Abiding by the guidelines is essential, he says. But “people get blasé, I hate to say it. After a while as microbiologists, you're like, 'This thing never bit me.'”

8. PSYCHIATRIC RESEARCH

# Is Internal Timing Key to Mental Health?

1. Yudhijit Bhattacharjee

Light therapy and other interventions that target the body's biological clock could help treat depression and other mood disorders

Every year, after the colors of autumn faded from the trees and left barren branches to herald the winter, Herbert Kern would feel his mental skies darken. As the days shortened, the middle-aged materials researcher would retreat from almost all social interaction. The routine was so familiar to Kern's colleagues at Bell Labs in Murray Hill, New Jersey, that they would not expect much work from him during those winter months in the 1970s. The seasonality of Kern's depression was reflected in the pocket notebooks in which he kept a log of his life. “During the rest of the year, I could fill a notebook every 2 weeks; in the winter, it would take months,” recalls Kern. Neither the few approved antidepressants of the time nor lithium injections did anything to help.

Then, in the late '70s, Kern learned about research in animals showing that melatonin, a hormone regulated by the light-dark cycle of day and night, plays a role in controlling seasonal behaviors such as mating. Wondering if the hormone had something to do with his condition, he got in touch with psychiatrist and melatonin specialist Alfred Lewy at the National Institute of Mental Health (NIMH) in Bethesda, Maryland, who was wrapping up a study demonstrating that exposure to bright light during the night suppressed nighttime secretion of the hormone in normal humans. When Kern sank into depression the following winter—in December 1980—Lewy's team exposed him to a few hours of light in the dark mornings and evenings, trying to match the amount of natural light of a spring day. After a few days of the treatment, “I began to be bubbly again,” says Kern, who later continued the regimen at home. “It worked like magic.”

Kern's case, and 3 years of follow-up work, led researchers to identify winter depression as a psychiatric illness that subsequently came to be known as seasonal affective disorder. SAD has since been shown to afflict millions of people, primarily in the northern latitudes, and a recent analysis by the American Psychiatric Association (APA) in Arlington, Virginia, provided a strong endorsement for light therapy as a treatment. And yet it's not settled how light, or other interventions that target the circadian clock, helps people with SAD.

SAD provides the strongest evidence to date of a link between the biological clock—the body's 24-hour timekeeper—and mental health, a proof of principle that circadian rhythms that are out of sync could underlie some mood disorders. But there is increasing evidence that circadian disturbances are involved in other common mental ailments such as bipolar disorder and more obscure ones such as a syndrome in which people compulsively eat at night. In recent years, psychiatrists working with small groups of patients have shown that correcting abnormal circadian rhythms—through exposure to light, melatonin pills, or even sleep deprivation—can help treat some of these disorders and can also benefit patients with neurodegenerative illnesses such as Alzheimer's. Some drug companies are even taking heed. “The circadian model is clearly beginning to bear fruit,” says David Avery, a psychiatrist at the University of Washington School of Medicine in Seattle. “It is logically getting extended beyond SAD and should lead to better treatments for a number of psychiatric disorders.”

When Kern contacted Lewy at NIMH, scientists already knew that all mammals have a master clock in the brain's suprachiasmatic nucleus, which regulates the waxing and waning concentration of numerous hormones and proteins in the body over an approximately 24-hour cycle. They also knew that the rhythms of many of these body chemicals—including melatonin, secreted by the pineal gland during darkness—were synchronized to the light-dark cycle of the environment. In treating Kern with light, the NIMH researchers—led by Lewy and his senior colleague, Thomas Wehr—simply simulated the earlier dawn and later dusk of spring, hoping that by shortening the duration of melatonin secretion, they'd lift Kern out of his depression.

Although it worked—and has since proven effective in treating many other cases of SAD—Wehr and Lewy formed different opinions about light therapy's mechanism. Wehr grew convinced that the antidepressant effect was a result of the artificially lengthened daytime, which led to less melatonin secretion and presumably had downstream effects leading to an improvement in the patient's mood. Lewy instead came to believe that the effect was due to the resetting of the patient's circadian clock, not the overall duration of melatonin production. In most SAD patients, he argued, the depression was the result of circadian clocks being out of sync with respect to the sleep-wake cycle, like a chronic form of jet lag. The theory has become known as the phase-shift hypothesis.

Last year, researchers led by Lewy—who has been at Oregon Health and Science University in Portland since 1981—presented the strongest evidence to date for this theory. Rather than using bright lights to reset the circadian clock, Lewy and his colleagues gave SAD patients melatonin pills. (The body's melatonin rhythm is tightly coupled to rhythms of other hormones such as cortisol and serotonin, and researchers have established that administering melatonin is a way to shift all of those rhythms en bloc.) People normally start secreting melatonin a couple of hours before bedtime to prime the body for sleep, so administering the hormone in the afternoon should advance a patient's circadian clock relative to his sleep-wake cycle. If given in the morning, it should have the opposite effect.

By making patients stick to their regular sleep times, the researchers ensured that their sleep-wake cycle remained constant throughout the study. After 3 weeks, they found that SAD patients whose circadian clocks normally lagged behind their sleep-wake cycle did better when they received afternoon melatonin and worse when they were given the hormone in the morning. The treatments had the opposite effects on those whose cycles were shifted the other way. Lewy points out that the treatments increased the duration of melatonin production, yet patients improved when their cycles were brought into sync. “If Tom [Wehr] was right, these people should have gotten worse,” he says. Lewy notes that the melatonin results are consistent with previous studies showing that morning light is significantly better at treating SAD than evening light (which corresponds to there being a higher proportion of phase-delayed rather than phase-advanced individuals among SAD patients).

Wehr, who retired from NIMH in 2003 and is now a practicing psychiatrist in Bethesda, Maryland, remains unconvinced. He points to animal studies showing that morning light brings about a quicker end to melatonin secretion without really affecting the hormone's onset time in the evening. It's possible, he argues, that afternoon melatonin led Lewy's patients to stop secreting the hormone a lot earlier than normal the following morning, in effect shortening the length of their melatonin production. To settle the question, Wehr says, researchers would need to keep a continuous track of the patients' 24-hour melatonin profile.

Lewy's hypothesis does not rule out the possibility that additional mechanisms are involved in light's antidepressant action. Some studies have shown, for example, that exposure to sunlight can increase brain levels of serotonin—a neurotransmitter associated with well-being—and Lewy says it's possible that serotonin is related to circadian alignment. To get a full mechanistic account of the clock's role in mental health, researchers still need to understand what cellular events are triggered when out-of-sync rhythms are snapped back into phase with each other, and by the same token, what happens in the brain when rhythms go awry—as they do even in healthy individuals who are jet-lagged.

A better understanding of these mechanisms could shed light on disorders beyond SAD, for abnormal circadian rhythms are turning out to be a factor in a number of other mental illnesses. Two years ago, in Chronobiology International, Vishwajit Nimgaonkar and his colleagues at the University of Pittsburgh Medical Center in Pennsylvania reported that among 75 patients with bipolar disorder, internal biological clocks—as measured by a questionnaire probing activity and sleep patterns—tended to be disturbed in comparison to those of a set of normal individuals.

And in two ongoing studies, researchers led by Anna Wirz-Justice of the Centre for Chronobiology at the Psychiatric University Clinic in Basel, Switzerland, are finding abnormal circadian rhythms in schizophrenic patients and in patients with borderline personality disorder. (The preliminary results from the studies were presented at the annual meeting of the Society for Light Treatment and Biological Rhythms in Copenhagen, Denmark, in June.) Also this summer, at a meeting on biological clocks and rhythms at Cold Spring Harbor Laboratory in New York state, psychiatrist Namni Goel of the University of Pennsylvania reported that many 24-hour hormonal rhythms in patients with night eating syndrome were either advanced or delayed with respect to the sleep-wake cycle.

Some researchers suspect that defects in the gears of the body's biological clock, caused by genetic mutations, will be shown to play a role in mental health problems. They point to studies such as one reported last year by Colleen McClung and her colleagues at the University of Texas Southwestern Medical Center in Dallas. The researchers created mice missing the Clock gene—which encodes a key protein in the machinery of the circadian system—and found that the animals showed manic behaviors, becoming hyperactive and keener to take risks. Expressing the CLOCK protein in the animals' midbrains restored behavior of the mutant mice to normal, McClung and her colleagues further reported in the 10 April issue of the Proceedings of the National Academy of Sciences this year.

If disrupted circadian rhythms contribute to mental illnesses other than SAD, those conditions could also benefit from light therapy. Indeed, researchers have begun testing this idea in small groups of patients, and they say the results look promising.

Nearly 200 people with Alzheimer's disease, spread across 10 homes for the elderly, are now helping researchers test whether light therapy can alleviate some symptoms of the fatal neurodegenerative disease—one of which is disturbed sleep-wake rhythms. Psychiatrist Eus J. W. van Someren of the Netherlands Institute for Neuroscience in Amsterdam and his colleagues have installed bright light fixtures in the lounges of six of the homes; at the remaining sites, they installed similar but fewer lights to set up the lighting equivalent of a placebo. Van Someren says that the unpublished preliminary results, based on more than 4 years of data, show that bright light improved the sleep-wake rhythms of patients. He claims the data also show that it slowed their cognitive decline, hinting that the disturbed circadian rhythms were a partial cause.

Light and melatonin are not the only interventions that researchers are using in their attempts to treat mental disorders by tinkering with the circadian clock. Francesco Benedetti, a psychiatrist at the San Raffaele Scientific Institute in Milan, Italy, has spent the last decade studying the antidepressant effects of total sleep deprivation—a strategy discovered by chance in the 1960s when German clinicians observed significant improvement in a depressed patient who had spent the night biking. Although how the technique works is poorly understood, psychiatrists routinely use sleep deprivation to produce a rapid emotional lift in deeply depressed patients, including those hospitalized after a failed suicide attempt.

Benedetti and others have shown that this dramatic effect, which invariably vanishes after a day, can be sustained for several weeks by using light therapy to shift the patient's sleep-wake cycle in the days that follow. The idea again is to bring the circadian rhythms back in alignment. The researchers have reported, in a study published in The Journal of Clinical Psychiatry in 2005, that combining light therapy with initial sleep deprivation can effectively treat bipolar patients. In a more recent study involving 55 bipolar patients, presented at the Cold Spring Harbor meeting by University of California, Irvine, psychiatrist Joseph Wu, those who received a treatment package including antidepressant medication, light therapy, and sleep-wake adjustment following total sleep deprivation did significantly better than those who only received medication.

## Witch healers?

Such studies seem to be making an impression on the broader psychiatric community. In 2005, a group set up by APA to examine the efficacy of light therapy concluded from a meta-analysis of published literature that the treatment significantly reduced depression symptoms in SAD, as well as in other mood disorders. The group, led by psychiatrist Robert Golden, now at the University of Wisconsin School of Medicine and Public Health in Madison, declared in April 2005 in The American Journal of Psychiatry that “the effects of light therapy are comparable to those found in many antidepressant pharmacotherapy trials.” However, the authors lamented the relatively small number of studies that met their criteria for the analysis—only 20 out of a total of 173 that were initially identified—and they noted that “additional randomized, controlled trials with appropriate numbers of subjects are needed.”

Until even a few years ago, “people looked at us as if we were some kind of strange witch healers,” says Benedetti, who began combining light therapy with sleep deprivation in the 1980s. Still, with recent data showing that drugs do not significantly help up to 40% of patients with mood disorders, he says, “there is a growing interest in chronobiological methods of treatment.”

Unfortunately, proponents of such methods say, funding has been hard to come by, in part because of the perception that effective antidepressants are available. Michael Terman, a psychiatrist at Columbia University, recalls that one grant application turned down by the National Institutes of Health contained this comment: “Why do we need a new antidepressant modality when we already know that drugs work?” The proposed work, a randomized trial testing light therapy in pregnant women with depression, is now being funded by the Swiss National Science Foundation.

“The pharma-driven model is so strong that it is difficult to win support for studying anything that does not involve drugs,” says Wirz-Justice.

Nonetheless, the French pharma company Servier has patented a melatonin agonist called agomelatine that is now undergoing clinical trials in Europe and in the United States as a treatment for depression. Merck has also set up a research group to look into the circadian basis of mood and sleep disorders in hopes of developing more effective drugs. “As therapies go, it would be far easier to pop a pill than carry around a fluorescent bulb,” says Anthony Gotter, a member of the group.

Kern, who is now practically blind from macular degeneration, would welcome a pill substitute. He says that light therapy became less and less effective for him over the years as his eyesight faded. “Now I can hardly see, and all hell has broken loose,” he says with surprising cheer in his voice. “I have had periods of depression lasting over a year, and highs lasting as long. I think my clock is just running freely, without any control by the environment. I don't know when I'm going to feel what.”

9. ECOLOGY

# Can Palm Oil Plantations Come Clean?

1. Richard Stone

Under fire for their poor environmental record, makers of the world's top vegetable oil are turning to scientists for advice on how to make their industry sustainable

TELUK INTAN, MALAYSIA—A canary-yellow machine lumbers onto a fallow oil palm field and, with a roar of its motor, rips into a pile of fronds and shavings of dead trunks. As plantation operators and scientists observe the mulching process, their guide, Cheriachangel Mathews, a senior manager at United Plantations' Jendarata Estate, warns that the group has been infiltrated. “We have a journalist with us,” he says. “I want him and all of you to know that nothing here—nothing—is wasted.”

Mathews has good reason to be concerned about the take-home message. With prices soaring, palm oil, Malaysia's number-one crop, has recently surpassed soybean as the top-selling vegetable oil in the world. Oil squeezed from palm fruit bunches is an ingredient in myriad products, from ice cream to soap, and it is being touted as a biofuel that can stem reliance on fossil fuels. But the industry has been taking a mulching in the press. Environmental groups have accused plantations of razing forests to plant the lucrative crop and slaughtering orangutans that pilfer and eat the fruit.

Hoping to turn over a new frond, the oil palm industry is now endeavoring to demonstrate its sustainability. It faces an uphill battle. A just-completed review by three dozen academics details species declines pinned on the oil palm, a native of West Africa that has become a dominant feature of Southeast Asia's landscape. It is an “unavoidable fact that the replacement of diverse tropical forest with an exotic monoculture significantly impacts biodiversity,” states the Biodiversity and Oil Palm Briefing Document. It will be presented at a gathering in November of the Roundtable on Sustainable Palm Oil (RSPO), in which industry officials, scientists, and other parties are hammering out a voluntary certification scheme for minimizing harm to the environment.

Scientists and like-minded industry insiders hoping to curb destructive growth may get help from the market. Rising palm oil prices are strangling demand for palm as a biofuel, Edgare Kerkwijk, managing director of the BioX Group, a renewable-energy company in Singapore, told the International Palm Oil Congress in Kuala Lumpur late last month. That's bitter news for companies in Southeast Asia that have been racing to ramp up capacity to process palm into biodiesel. With crude palm oil now topping $700 per ton, “we believe that palm oil is not a long-term biofuel,” Kerkwijk said. The industry, nevertheless, is riding high. According to the Food and Agriculture Organization of the United Nations (FAO), global palm oil production last year was 37 million tons, 85% from Indonesia and Malaysia. Palm oil yields—2.8 tons per hectare, on average—are seven times those of soybean oil, according to FAO. Aiming for even higher yields, the Asiatic Centre for Genome Technology in Kuala Lumpur and Synthetic Genomics, a company in Rockville, Maryland, founded by J. Craig Venter, in July announced a partnership to sequence and analyze the oil palm genome. Higher yields are vital to an industry looking to clean up its act. Seen from the air, peninsular Malaysia is a patchwork of settlements and plantations interspersed with forest; in 2003, the peninsula had more than half of the country's 3.7 million hectares of oil palm. Malaysian officials maintain that plantations are now allowed to expand only onto existing agricultural fields or degraded land. Indonesia is a different story. There, renegade plantations fuel expansion through timber sales. “At the state level, there are no clear limits on plantation growth,” says Reza Azmi, director of Wild Asia, a company in Kuala Lumpur that is advising plantations in both countries on how to limit their environmental footprint. RSPO was formed 5 years ago to turn the positive environmental record of outfits such as United Plantations into a competitive advantage through the certification of “sustainable palm oil.” To bolster this effort, a network of researchers drew on a wealth of data to assess the impact of plantations on biodiversity. An advanced draft of the document provided to Science paints a grim picture. The authors, led by Emily Fitzherbert of the Zoological Society of London, summarize research documenting shifts in biodiversity in and around plantations. In Sumatra, for example, less than 10% of birds and mammals found in primary forests live in plantations, and more than 75% of bat species were lost; in Thailand, 41 bird species were found in plantations, compared to 108 species in nearby tropical forests. “Plantations need to accept that oil palm is not compatible with biodiversity,” says report coauthor Matthew Struebig of Queen Mary, University of London, U.K. “Environmental groups and scientists need to work with, not against, the industry to help them minimize this impact.” The document delivers a clear bottom line to RSPO: “The most immediate and important action needed to prevent further biodiversity loss is to ensure that oil palm expansion does not contribute to deforestation.” The report also highlights how proactive management can reduce species losses, for example by salvaging native stands inside plantations. Wild Asia is working with plantations on plans to link fragments into “natural corridors” and set aside 50 of every 2000 hectares for forest regeneration. “Two years ago,” says Azmi, “this discussion would never have happened.” 10. RESEARCH IN JAPAN # Big Winners, Big Expectations 1. Dennis Normile Five groups have been awarded decade-long grants in a drive to win global attention and draw international talent TOKYO—Immunologist Shizuo Akira is indisputably at the top of his field. For 2 years running, the Osaka University professor has been Thomson Scientific's “Hottest Researcher” for authoring the most highly cited papers in his field. But Osaka has not won recognition as a leading world center for immunology research; Akira fears the university may even be in danger of falling behind. Advancing technology “makes it very difficult for a single laboratory” to create an international buzz, he says: “What's needed is to accumulate a research team and get a big grant.” He has just gotten a very big grant; Japan hopes the international buzz will grow. Akira's center is one of five selected to receive in the neighborhood of$12 million per year for 10 years under a World Premier International Research Center Initiative sponsored by Japan's Ministry of Education. The grants, which must be supplemented by the host institutions, are intended to take the winners to a new level of global prominence through generous, discretionary funding and support for internationalizing research. Akira hopes to lure leading Japanese and foreign immunologists to Osaka and, in particular, push into the nascent field of in vivo imaging of the cell-cell interactions that define immune response.

The grant program is an audacious bet by Japan's Ministry of Finance, which is out to make at least this handful of centers as widely recognized as the Massachusetts Institute of Technology's Media Lab or the U.K.'s Laboratory of Molecular Biology in Cambridge. “It's a visionary program,” says Matthew Mason, director of the Robotics Institute at Carnegie Mellon University in Pittsburgh, Pennsylvania. Mason was one of six foreign scientists on an international panel that reviewed 13 short-listed applications. The objective was to “pick groups already at the peak [of their field] and give them support to make them globally visible,” says Hiroshi Ikukawa, who is heading development of the program for the Ministry of Education.

Tohoku University in Sendai, for example, proposed creating an atom-molecule-materials center around its Institute for Materials Research, which is already one of the world's most prolific material science groups. Yoshinori Yamamoto, slated to direct the new center, says they hope to take their work on bulk glass materials to a new level by adding theorists and computational scientists. The University of Tokyo is partly building on the breakthrough studies of neutrinos done at its Super-Kamiokande Neutrino Observatory with a new Institute for the Physics and Mathematics of the Universe. Hitoshi Murayama, a theoretical physicist at the University of California, Berkeley, says they will bring together experimental observations, theory, and new mathematical approaches “to try to understand such basic questions as how the universe started and where it's going.”

View this table:

New horizons. Findings at the Super-Kamiokande Neutrino Observatory at the University of Tokyo (above) led to a grant for an international math and physics institute.

Global visibility has eluded Japan's universities and research institutes for a variety of subtle reasons. Norio Nakatsuji, a cell biologist at Kyoto University who will be heading its new Institute for Integrated Cell-Material Sciences, cites geographical isolation and the language barrier. So the initiative has set a target for each center to have 10% to 20% of its two dozen or so principal investigators (PIs) and 30% of an expected 200 research staff be non-Japanese. And “naturally, English should be the language of the centers,” says Nakatsuji.

Paul Weiss, a chemist at Pennsylvania State University in State College, who will be affiliated with the Tohoku center, says, “Another [problem] is the hierarchy typical in Japanese scientific institutions.” To counter this, Weiss says, “we are making a concerted effort to encourage creativity and independence among young scientists.”

And Masakazu Aono, director of the new Center for Materials Nanoarchitectonics at the National Institute for Materials Science in Tsukuba, says that probably because of rigid academic structures, “Japanese scientists have not been good at interdisciplinary collaboration.” His center will bring a range of specialists together to study nanoscale structures to create new types of alloys and microelectronic devices as well as organic and biological materials.

Weiss, for one, is envious. “Where can we ask for resources in the U.S. to go after a 10-plus-year problem? What mechanism lets us put together a team of the top people from all over the world?” he asks.

Still, some researchers are concerned about the depth of commitment. “There is no tenure [in this program],” notes Murayama, who will head the new center at the University of Tokyo. “So how do we make the jobs at this institute competitive” with the best permanent jobs elsewhere? he asks. And there are questions about the involvement of the non-Japanese PIs. Most, including Weiss, will likely maintain their current positions, devoting just a percentage of their efforts to the centers.

Program backers hope the part-time presence of leading foreigners plus full-time Japanese scientists will attract younger researchers of all nationalities on a full-time basis. Kyoto's Nakatsuji says they have plans for “superpostdocs,” under which select newly minted Ph.D.s could be given the money to independently run a small group, complete with technicians and graduate students. Osaka's Akira hopes some of these young scientists will become world leaders—and stay in Japan.

11. TROPICAL DISEASES

# Hunt for Dengue Vaccine Heats Up as the Disease Burden Grows

1. Dennis Normile

As the number of cases reaches an all-time high, new techniques and an influx of research funds could mean this long-neglected disease will finally have a vaccine

For decades, Duane Gubler and other arbovirus experts have been warning about a looming dengue crisis. But dengue fever, transmitted most often by the bite of an infected Aedes aegypti mosquito, was often seen as an obscure, only occasionally fatal disease of tropical countries, and progress toward a vaccine and drugs to treat it has been slow.

Now, with cases exploding across Southeast Asia and the disease apparently becoming more virulent and spreading into new geographic areas, vaccine research is taking on a new urgency. “For 30 years, we've been saying a dengue vaccine might be available in the next 10 years,” says Gubler, a dengue expert at the University of Hawaii, Manoa, in Honolulu. “And now, finally, it seems we may be right about that.” Some long-running research is finally bearing fruit, says Gubler, and as dengue captures global attention, the pharmaceutical industry is boosting investment in both traditional and novel vaccine technologies. The Bill and Melinda Gates Foundation in Seattle, Washington, has chipped in a \$55 million, 5-year grant to set the stage for phase III trials, which will help speed candidate vaccines to market.

A vaccine can't come a moment too soon. On average, fewer than 300,000 cases of dengue a year were reported to the World Health Organization (WHO) during the 1980s; since 2000, that number has exploded to 925,000. Because surveillance is poor, WHO estimates that the true number of dengue cases tops 50 million annually, including about 400,000 cases of dengue hemorrhagic fever (DHF), a severe and sometimes fatal form of the disease. In Southeast Asia, dengue is starting to rival malaria as a killer of children, and its economic impact is already greater. The spike in cases is driven by the collision of several trends, chief among them rapid urbanization and to some extent poverty. The warm, crowded cities of Latin America and Asia provide an ideal habitat for the main vector, A. aegypti, which breeds in stagnant water and likes to feed on humans in quick succession. Faced with record-setting outbreaks this year in Southeast Asia, WHO sounded the alarm on 23 July, calling on countries to dramatically step up mosquito control—for now, the only means of prevention—and improve patient care. But to date, most developing country governments have a dismal record of mounting and sustaining effective vector-control programs. This means, Gubler says, “it's going to continue to get worse until we have a vaccine.”

Dengue is caused by four closely related viral serotypes—dengue 1 through dengue 4—which are single-stranded RNA viruses spread primarily by the A. aegypti mosquito. The disease in humans ranges from mild to mortal. A week or so after infection, the typical patient suffers a rapid onset of fever with excruciating joint pain—dengue is called “breakbone fever” in some regions—and sometimes nausea and skin rashes. About 1% of cases progress to DHF, with internal bleeding that can lead to shock and death, although fluid replacement therapy usually saves those hospitalized in time.

Once they recover, patients are immune for life, but only to the dengue serotype that infected them. For poorly understood reasons, those subsequently infected with a second serotype are at far greater risk of progressing to DHF. Studies show that more than 90% of DHF patients had a previous dengue infection.

And the odds of DHF are increasing. The four serotypes used to be isolated geographically, making second infections rare. But, probably because of increased human mobility, now all four viruses often circulate in a region simultaneously. What's more, says John Ehrenberg, a WHO adviser on vector-borne diseases based in Manila, “the virus has changed genetically over the past 2 to 3 decades into more pathogenic strains.” This makes first infections more serious and second infections even worse. “A lot more cases are ending up in hospital because of complications,” Ehrenberg says.

## A shot in the dark

Since work began in the 1940s, vaccine developers have tripped over one stumbling block after another. Early on, researchers feared that someone vaccinated against one type of dengue might suffer the immune-enhancement response leading to DHF if later infected with a different serotype. Avoiding this response requires a tetravalent vaccine that provides roughly equal and lasting protection to all four dengue serotypes. But decades of research have shown that “it is hard to make a tetravalent vaccine so [that] there is simultaneous immunity against all four [viruses],” says Alan Barrett, a virologist at the University of Texas Medical Branch in Galveston. Ehrenberg adds that although other viruses also have different serotypes, only dengue provokes immune enhancement. Work has also been stymied by the difficulty of growing the virus in culture and by the lack of an animal model. Monkeys infected with dengue produce antibodies but don't really suffer from disease, limiting their experimental value.

Working separately, researchers at Mahidol University in Bangkok and at the Walter Reed Army Institute of Research (WRAIR) in Silver Spring, Maryland, were the first to report in the 1990s significant progress toward a tetravalent dengue vaccine. Both groups set their sights on a live attenuated vaccine, in which a live virus is weakened by repeated replication in a cell culture. Both groups struggled to find the right level of attenuation at which the virus is strong enough to trigger an immunogenic response but weakened enough so that it cannot cause illness. Without an animal model, the researchers had to develop techniques, such as how the virus affected different cell cultures, by which to judge attenuation. And they had to develop a different vaccine for each dengue serotype, test it in humans, combine them, and go through human trials again.

By the early 1990s, after more than a decade of development, the Mahidol group had combined attenuated strains of dengue 1, 2, and 4 into a vaccine that “got good results,” says Sutee Yoksan, a virologist who heads Mahidol's Center for Vaccine Development. But when they added their dengue 3 vaccine to the cocktail, things went awry. Some volunteers got sick from the dengue 3 component, which also interfered with the production of antibodies, leaving those inoculated with little or no protection against the other three serotypes. The vaccine was licensed to what is now Sanofi Pasteur in Lyon, France, where researchers tried genetically weakening the dengue 3 component. But trials of this monovalent dengue 3 vaccine in 2002-2003 still sickened volunteers, and Sanofi Pasteur has given up on the vaccine. In Bangkok, Yoksan is still screening dengue 3 viruses for another attenuation candidate. “If we can solve the dengue 3 problem, we will have a good vaccine,” he says—but he won't predict how long it might take.

The group at WRAIR also had trouble with its dengue 3 virus but eventually attenuated it. GlaxoSmithKline (GSK) has taken over clinical development of the vaccine and hopes to start a phase II field trial next year. Several thousand people in vaccinated and control groups will be tracked for 1 to 2 years to compare rates of infection.

Bruce Innis, a GSK physician and virologist who previously worked on the vaccine at WRAIR, says that except for a very limited trial in the early 1960s, this will be the first time a dengue vaccine will be tested for actually preventing illness, as opposed to simply measuring neutralizing antibody production. With most diseases, mice or monkeys can be challenged with infection to determine the efficacy of a candidate vaccine, but that doesn't work for dengue. In human trials so far, researchers have inferred the degree of efficacy by measuring the production of neutralizing antibodies in response to vaccination in human volunteers. “But we don't know how much antibody you need to have in order to conclude that someone is protected,” says Innis. The GSK field trial will provide the first data directly relating antibody production to disease protection.

Because of the slow progress with live attenuated vaccines, researchers have been working on alternatives. The furthest along is a chimeric vaccine that uses a yellow fever vaccine virus as a backbone but replaces several key structural genes with dengue counterparts. The technique was pioneered at Saint Louis University in Missouri, further developed by the company Acambis in Cambridge, Massachusetts, and Cambridge, U.K., and finally licensed to Sanofi Pasteur, which now has a tetravalent vaccine in phase II clinical trials in adults and children.

Researchers say there is merit in both approaches. One advantage of chimeric vaccines, says Gubler, is that researchers can genetically manipulate them to fine-tune the degree of attenuation. A downside, adds Innis, is that such recombinant vaccines have just a few of the wild-type dengue genes, whereas the live attenuated vaccines have all 10 genes for each component, possibly making them more efficacious. “The real question is 'What works?'” says Barrett, who expects some answers to come out of the ongoing trials.

As these and other candidate dengue vaccines (see table) wend their way through early-stage clinical trials, the Pediatric Dengue Vaccine Initiative (PDVI) in Seoul, South Korea, is using the Gates grant to ready field sites for the large-scale phase III trials that will be needed to license a vaccine. Even before trials start, baseline data on field trial sites are needed, which require laboratories and staffs conducting ongoing surveillance of the dengue viruses in circulation and collecting epidemiological data such as infection rates. All vaccines face this hurdle, but dengue's is a bit higher because researchers must distinguish and track the four dengue viruses. PDVI Director Harold Margolis says “it will be difficult” to show efficacy against all four serotypes with trials at just one site, because one virus usually predominates in a region. Trials may have to be done at multiple sites, although researchers and regulators are still pondering the best approach. PDVI is also working on standardizing laboratory diagnostic protocols and clinical case definitions to support clinical trials.

Gubler, who chairs PDVI's board of counselors, says, “This saves the manufacturers a lot of time and a lot of money [because] they don't have to develop these field sites themselves.” He believes this logistical help has encouraged smaller firms with novel approaches to take up the dengue vaccine challenge and thinks it may shave 3 years off development time. Now all they need are some phase III candidates, which Gubler and others predict should come along well within the next 10 years.