T. b. gambiense, which occurs in central and western Africa, is far and away the larger problem, accounting for 95% of reported cases and causing a chronic illness that can last for months or years without major symptoms. Nevertheless, most research into the disease has focused on the subspecies found in eastern Africa, T. b. rhodesiense, because this organism is easier to culture in the laboratory. Unfortunately, the two parasites employ different strategies for evading the immune system, so research on one doesn’t necessarily translate to the other.

Sleeping sickness is treatable, but new drugs are desperately needed. For decades, the main medication used for late-stage sleeping sickness was melarsoprol, a compound derived from arsenic that wipes out the parasite but also kills 3–10% of people on the drug. Last year, the World Health Organization introduced a combination of two compounds—eflornithine and nifurtimox—that is safer than melarsoprol, but the treatment doesn’t work against T. b. rhodesiense. And because eflornithine must be given intravenously for a week, the combination

other primates to resist the parasite. Yet it is still unclear whether they can leverage their newfound knowledge to curb the disease.

Sleeping sickness (also known as African trypanosomiasis) occurs in sub-Saharan Africa wherever infected tsetse flies are found. These brown insects—about the size of houseflies—bite humans, as well as domestic animals and wild game, transmitting three different subspecies of the Trypanosoma brucei parasite from their salivary glands into the host’s blood. Humans are immune to one of these subspecies, T. b. brucei. But the other two, T. b. rhodesiense and T. b. gambiense, are often lethal.

The first symptoms of infection are mild—bouts of fever, headaches, joint pain and itching. By the time people realize they are sick, the parasite has often already made its way to the brain. There it wreaks havoc, causing rapid mood swings, confusion and poor coordination. A hallmark of the advanced form of the disease is an inverse sleep-wake cycle—insomnia at night, drowsiness during the day—hence the disease’s name.

Jayne Raper, her long brown hair swept back into a bun, leans forward to look through the twin lenses of a benchtop microscope. The slide below them contains hundreds of wriggling parasites. These are African trypanosomes—not the variety that causes sleeping sickness in people, but a very close cousin that infects cattle and other animals. These single-celled protozoans just came out of the freezer, but the microscope’s hot light makes their knife-like bodies thrash and twist. “They’re beautiful, aren’t they?” she muses.

‘Beautiful’ may not be a word that most researchers would use to describe this deadly parasite. But Raper, a biochemist at New York University School of Medicine, is mesmerized by the microbe and the possibility of thwarting its defenses to combat sleeping sickness, a plague that afflicts an estimated 30,000 people living in Africa each year. Over the past two decades, Raper and other basic scientists have learned an astonishing amount about how this tiny organism evades the human immune system, and they have uncovered genetic mutations that allow humans and

For most people, a single bite from a parasite-infected tsetse fly can trigger a slow, agonizing and sometimes fatal disease known as African sleeping sickness. But new research shows that some people, as well as baboons and other great apes, are naturally resistant to infection. Cassandra Willyard awakens to the possibility of using existing immunity to engineer new therapies and transgenic livestock.

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is still difficult to administer in the remote parts of Africa hardest hit by the disease.

Raper and her colleagues hope their work will open the door to new ways of preventing the disease. Whereas most investigators are searching for ways to block infection in people, Raper is approaching the problem from a different direction—the barn. Many of the same species that infect people also infect livestock. By engineering a cow that can resist the parasite, she hopes to reduce the number of infected hosts and curb the spread of the disease in both cattle and people.

The killing factorRaper first became interested in sleeping sickness in the 1980s while working as a lab technician for biochemist Mervyn Turner (now with Merck) at the now-defunct Molteno Institute of Biology and Parasitology at the University of Cambridge, UK. At the time, scientists knew that human blood possessed some kind of trypanosome-killing factor that wiped out trypanosome subspecies such as T. b. brucei, but they didn’t understand how this innate protection worked or why other subspecies were immune to the toxic factor. Because this mysterious molecule seemed to bust up the pathogen’s cells, it became known as ‘trypanosome lytic factor’, or TLF. And, over the next two decades, Raper and two competing scientists began to unravel why this unique molecule works against some trypanosomes but not others.

TLF is a type of high-density lipoprotein (HDL), the ‘good cholesterol’ that disposes

of artery-clogging plaques. But TLF has two components not found in other HDLs—which are not single molecules but rather assemblies of many proteins and lipids. The first, identified in 1995 by Stephen Hajduk, a molecular biologist now at the University of Georgia in Athens, is haptoglobin-related protein (Hpr). Hajduk saw that Hpr binds hemoglobin, and he proposed that, together, these proteins could be toxic, triggering oxidative damage. In his landmark experiment, Hajduk and his colleagues blocked the protein’s activity by adding Hpr-specific antibodies to a sample of TLF. They then added T. b. brucei and saw that the more antibody included in the assay, the fewer parasites died1.

Across the Atlantic, Etienne Pays of the Free University of Brussels in Belgium, was also working to understand what makes TLF so lethal. In the late nineties, Pays uncovered the mysterious component in T. b. rhodesiense that allows it to neutralize TLF, which led him to TLF’s other key component: apolipoprotein L1 (ApoL1). In 2000, Pays observed that human serum lacking ApoL1 lost the ability to kill trypanosomes. When Pays’s team exposed T. b. brucei to recombinant ApoL1, they saw that it killed the parasite2. Thus, Pays posited that ApoL1, not Hpr, is the key trypanosome-killing factor.

Raper, meanwhile, had begun hunting for the components of TLF in baboon blood. Baboons, like some other primates such as mandrills and sooty mangabeys, are immune to all African trypanosomes, and Raper

thought these proteins might have a crucial role in their ability to resist infection.

Her search first yielded Hpr. In 2004, she and her colleagues showed that antibodies

against the protein seemed to neutralize the trypanosome-killing ability of baboon blood in vitro3. But when her lab inserted the baboon version of the gene encoding Hpr, the researchers found that the mice remained susceptible to the parasite4. After that letdown, Raper shifted her attention to ApoL1.

Raper’s previous antibody-based searches for ApoL1 in baboon blood had failed to detect the protein because,

Raper realizes in hindsight, baboon ApoL1 is only around 60% similar to the human equivalent. But when she turned to more sensitive alternative methods, such as mass spectrometry, Raper saw that ApoL1 was there all along. In 2009, her lab described the baboon gene that codes for ApoL1 and showed that inserting this gene into mice provided complete protection against T. b. rhodesiense infection4.

And it isn’t just baboons and other monkeys that carry a protective version of the APOL1 gene. Last summer, Martin Pollak, a geneticist who studies inherited kidney diseases at the Beth Israel Deaconess Medical Center in Boston, teamed up with Pays to show that two rare alleles of APOL1 found in some humans can also kill T. b. rhodesiense5. People who carried two copies of the defective gene were more likely to have kidney disease. The fact that these mutations occur only in people of African descent suggests that they evolved as a countermeasure against African trypanosomes.

Tsetse fly stage Human and other mammalian host stage

Tsetse fly takes a blood meal

(trypanosomes are injected)

Tsetse fly takes a blood meal

(trypanosomesare ingested)

Injected trypanosomes

multiply in various body fluids,

including blood

Bloodstreamtrypanosomes

travel to the fly’smidgut and thensalivary glands

“One milliliter, maybe less, should be enough to completely cure people having trypanosomes in their blood.”

Sleeping sickness cycle: The infection cycle of the vectors that cause African trypanosomiasis.

Rapier wit: New York University’s Jayne Raper.

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Raper, Pays and Hajduk all agree that ApoL1 is important, but they can’t seem to come together on the specifics. All three researchers permit that when a human becomes infected with T. b. brucei, Hpr (bound to hemoglobin) attaches to a receptor on the surface of the parasite, allowing TLF to gain entry into the cell. Once inside, the molecule travels to the parasite’s gut-like sac—the ‘lysosome’—where TLF gets broken down to its constituent parts. In this acidic environment, ApoL1 becomes activated, binds the lysosome’s membrane and forms tiny pores.

That much the researchers can agree on. But exactly how TLF kills trypanosomes has been a matter of fierce debate. “I can, from personal experience, tell you that it can be very tense when people from these three labs get together,” says James Bangs, a cell biologist who studies trypanosomes at the University of Wisconsin–Madison, adding that resolving the points of disagreement will “require a little less passion and a little more objectivity.”

Raper acknowledges that the groups have their disagreements but says the rest of the field gets a kick out of it. At conferences, “it’s one of the highlights,” she jokes. Hajduk points out that the groups actually agree on many important points. “It’s unfortunate that this whole field comes down to what appears to be this huge controversy,” he says.

The main points of contention boil down to how exactly Hpr and ApoL1 interact to kill the parasite. In 2007, Pays showed that ApoL1 forms pores that allow chloride ions and water to flow into the lysosome, which causes the parasite to swell, burst and die6. Raper agrees that ApoL1 is the toxin. In her mouse model,

Hpr alone did not protect the mice at all, she says. However, neither Raper nor Hajduk has been able to recreate the swollen lysosomes that Pays sees in his lab. Raper thinks ApoL1 must form pores in the parasite’s membrane as well.

Hajduk concedes that ApoL1 forms pores, but he isn’t convinced that it is the sole toxin. He proposes that the Hpr-hemoglobin combination causes oxidative damage in the lysosome that leads to the breakdown of the membrane. This damage, combined with the pores formed by ApoL1, he says, causes the parasite’s untimely demise7.

Further fueling the controversy, Hajduk recently published a paper explaining how T. b. gambiense is able to evade TLF. In September, he and his colleagues reported that the cell surface receptor that helps TLF gain entry into the parasite is not functional in this subspecies8. Pays contends that Hajduk’s results are incorrect and hints that his lab has unpublished evidence for a different mechanism, although he declined to provide specific details.

Have a cow, manArguments aside, all three research teams have the same goal. By gaining a better understanding of how African trypanosomes interact with their hosts, they hope to find new ways to prevent and treat the scourge of sleeping sickness.

Pays suggests that physicians may be able to use serum from people with the rare APOL1

mutation that he and Pollak recently identified to treat sleeping sickness in the field. “One milliliter, maybe less, should be enough to completely cure people having T. b. rhodesiense in their blood,” he says. Meanwhile, Raper suggests it may be possible to manufacture a version of human ApoL1 that contains the protective mutation found in baboons. And because the protein would be so similar to human ApoL1, it might not even trigger an immune reaction, she notes.

But experts who work in drug development and public health are skeptical that these approaches would be effective in the fight against sleeping sickness. For starters, notes Alan Fairlamb, a biochemist who investigates drugs targeted at trypanosomes at the University of Dundee, UK, most cases of sleeping sickness are caused by T. b. gambiense, not the T. b. rhodesiense subspecies that most researchers have focused on. In addition, Fairlamb points out that sleeping sickness occurs in remote, poverty-stricken areas where hospitals may not have the ability to administer treatments such as expensive recombinant proteins, which must be kept cold.

Delivering a serum therapy would not be easy either, says Raper, who has tested this approach in mice. Patients would probably need multiple shots, which means a more complicated regimen, she notes. What’s more, any therapy that requires intravenous injections poses delivery problems in the field. “If you want to succeed in trypanosomiasis control, you need to have oral treatment,” says Olaf Valverde, a medical manager and expert in sleeping sickness at the Drugs for Neglected Diseases initiative in Geneva. “Forget everything else.”

Rather than focusing on people directly, Raper thinks that she can curb the spread of sleeping sickness by preventing disease

Hard work pays off: Etienne Pays (right) and his colleagues discuss their latest findings.

Trippy trypanosome: A dividing bloodstream parasite.

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that confer antibiotic resistance won’t be easy. But she and her colleagues—she calls them her “cowboys”—should soon know whether developing a healthy cow that can resist the parasites is possible. Her funders want the group to deliver the transgenic cattle in three years. Raper says her team should have a cow embryo ready for transplantation in six months to a year. The breed even has a name—Chuma—which means ‘strong’ in Swahili.

Creating a mouse that can resist trypanosomes has been the highlight of Raper’s career. But building a resistant cow would be “the most exciting thing I’ve ever done,” she says. “I just hope we can do it.”

Cassandra Willyard is a science writer based in Brooklyn, New York

1. Smith, A.B., Esko, J.D. & Hajduk S.L. Science 268, 284–286 (1995).

2. Vanhamme, L. et al. Nature 422, 83–87 (2003).3. Lugli, E.B., Pouliot, M., Molina-Portela, P., Loomis,

M.R. & Raper, J. Mol. Biochem. Parasitol. 138, 9–20 (2004).

4. Thomson, R., Molina-Portela, P., Mott, H., Carrington, M. & Raper, J. Proc. Natl. Acad. Sci. USA 106, 19509–19514 (2009).

5. Genovese, G. et al. Science 329, 841–845 (2010).6. Vanhollebeke, B., Lecordier, L., Perez-Morga, D.,

Amiguet-Vercher, A. & Pays, E. J. Eukaryot. Microbiol. 54, 448–451 (2007).

7. Shiflett, A.M., Bishop, J.R., Pahwa, A. & Hajduk, S.L. J. Biol. Chem. 280, 32578–32585 (2005).

8. Kieft, R. et al. Proc. Natl. Acad. Sci. USA 107, 16137–16141 (2010).

9. Batchelor, N.A. et al. PLoS Negl. Trop. Dis. 3, e563 (2009).

works as part of a public-private partnership campaign called Stamp Out Sleeping Sickness, “people are reluctant to accept anything transgenic,” she says.

Welburn acknowledges that cows are important—her research shows that the movement of infected livestock may be

responsible for the spread of the disease from endemic to nonendemic regions in Uganda9. But she argues that introducing transgenic cattle is neither the smartest nor the most cost-effective way to tackle the disease, noting that “we have a very effective

way of getting rid of those parasites in domestic livestock. You just have to give them an injection of a trypanocidal drug. That’s quite a lot easier than proposing transgenic animals for Africa.”

Valverde takes a more positive view. Not being able to have cattle “is clearly a huge handicap” for people in areas affected by sleeping sickness, he says. So if Raper and her colleagues could build a transgenic cow, “it may bring revolutionary change,” he says. Still, he worries that putting the transgene into cattle could lead to health problems in the cows—for instance, kidney disease like Pollak and Pays observed in humans with a similar mutation.

Raper understands the obstacles. Introducing the mutant gene in the exact right spot in the genome without also adding genes

transmission in one of the parasite’s largest reservoirs—domestic cattle.

Cows are susceptible to some of the same trypanosomes as humans. So, by building a resistant cow, Raper and her colleagues hope to reduce the number of host organisms and, ultimately, the number of cases of sleeping sickness. Resistant cows would also give farmers in the ‘tsetse belt’ an additional source of food and income.

Raper first learned about this problem in 2006, when she attended a conference in Nairobi, Kenya and met with ranchers and scientists who were trying to breed resistant cattle. “I never really considered the economic impact of not being able to raise cows in the whole tsetse fly belt,” she says. Soon after that meeting, Raper discovered that baboon ApoL1 could protect mice from T. b. rhodesiense. If it can protect mice, she thought, why not a cow? “That’s when we knew that we could potentially make a transgenic animal,” she says. Last March, she and her colleagues received $2 million from the Bill & Melinda Gates Foundation and the US National Science Foundation to test her idea.

Yet, even if the project pans out, there might not be much demand for an engineered cow, notes Sue Welburn, a molecular epidemiologist at the University of Edinburgh, UK. In the rural communities of Uganda, where Welburn

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Until the transgenic cows come home: Researchers hope to engineer cattle like these in northern Uganda to resist the sleeping sickness parasite.

“A trypanosome-resistant cow may bring revolutionary change.”

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