The Littlest Killers

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Author: Erik Ness

In the spring of 1855, Father Edward Sorin, CSC, must have cast a troubled gaze upon the marshy land surrounding Saint Mary’s and Saint Joseph’s lakes. Only the year before a typhoid epidemic had devastated the Notre Dame campus, and now two early deaths in March suggested the fever would rise again. The likely cause, the University’s founder believed, was high lake levels caused by a dam on a neighboring farm. Malaria, cholera and yellow fever plagued Notre Dame’s early years, and Sorin blamed the over-full lakes. When the landowner reneged on negotiations to sell, Sorin famously took matters into hand and sent a half dozen men over to tear down the dam.

It would be a few more decades before Sorin’s instincts were confirmed by science. Swamp-born mosquitoes were a major culprit, lively vectors of malaria and yellow fever. But more than 150 years later, his solution still qualifies as state-of-the-art, particularly for malaria, which remains one of the world’s most intractable health problems. Up to 500 million cases occur annually, and the most virulent form—_Plasmodium falciparum_—takes more than 1 million lives each year. Some estimates put the yearly toll of malaria well beyond 3 million.

To make matters worse, the malarial parasite is increasingly drug resistant. In the same way, the mosquito vectors are adapting to insecticides and repellants. Environmental and social changes are expanding the disease’s range and compounding its impact. The technologies that work—insecticides, bed nets, habitat modification—are ancient concepts.

“There really hasn’t been anything new developed for dealing with most vector-borne diseases,” says Frank Collins of Notre Dame’s Center for Global Health and Infectious Diseases. A new movement aims to change that. With new tools, a renewed global commitment, and a fresh infusion of capital and strategic thinking, scientists at Notre Dame and beyond are working especially hard to make a target of malaria and other intransigent infectious agents.

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Notre Dame’s role is both next generation and back-to-basics. Researchers are using the genome—the high-tech, wholesale reading of the genetic code—to provide new clues to the basic biology. “Let’s just understand these organisms better,” says Collins. “If we can learn enough about these mosquitoes or sand flies or ticks, we might be able to devise an entirely new approach.”

For example: Redesign the mosquito.

George Craig, a leading mosquito researcher who worked at Notre Dame until his death in 1995, imagined using genetic engineering to simply eliminate the mosquito. This generation has a more subtle approach. Scientists already know how to reprogram mosquito DNA. They are confident that they can soon figure out what changes would render a mosquito “vector incompetent”—unable to carry deadly malaria or dengue fever. And they believe they can convince nature to incorporate this “improvement.” Their hardest job may just be convincing the public.

The book of genes

“The mosquito is both an elegant, exquisitely adapted organism and a scourge of humanity.” Those words capture the tricky problem of malaria and marked the beginning of a new era for vector biologists. They lead off the October 2002 publication in Science magazine of “The Genome Sequence of the Malaria Mosquito Anopheles gambiae.” It’s an understated title: Anopheles gambiae is the most important carrier of malaria in the world, and reading the genome opened new paths for malaria researchers. Nature published the genome of the malarial parasite Plasmodium falciparum that same month, raising hopes that the new science of genomics would quickly banish the ancient malady.

It was the kind of result that warranted celebrations, but the accomplishment was more start than finish. The published genomes hadn’t done much more than force open the library door. Once inside, the real work began. “It’s like walking into the Library of Congress without any kind of card catalog,” says Collins, a contributor to the decoding process.

Collins was an English graduate student before taking up mosquito biology, and he likens understanding the genome to reading a great novel. “At the most simplistic layer, you’re reading words. The words don’t necessarily mean a lot. They have some kind of general meaning: noun, verb, or a preposition or conjunction. When you put them together they form sentences and paragraphs, which themselves specify higher levels of meaning and complexity.”

So far, a lot of meaning escapes scientists. “We have a pretty good handle on the spelling,” he says. “We know the letters.” In A. gambiae researchers recognize probably 90 percent of the words. Each word is a gene, and it carries the instructions to make a single protein. The general meaning of probably about half of those words is known. Scientists can read some sentences. Occasionally they can even read several sentences together: How a dozen or more proteins all interact to do something. “But how do all 500,000 words in this book go together to make a novel that means something? That’s pretty far off.”

Decoding the genome was automated, enabled by fabulously expensive machines that break up long chains of DNA into readable segments. A computer then puts the whole puzzle together again.

Interpretation takes a bit more work, but comparison is critical. For example, A. gambiae is actually one of seven species that look identical under the microscope. A. gambiae prefers to bite humans, but another, A. quadriannulatus, prefers animals. Comparing the genomes might allow researchers to figure out why A. gambiae is so partial to humans. That insight might in turn allow the design of a better repellant or yield a target for genetic modification. To make this kind of comparison easier, researchers can use VectorBase, a new database of genomes for disease vectors directed by Collins. Already in its library is A. gambiae. The genome of another mosquito, Aedes aegypti, will be completed this fall; the work is being coordinated at Notre Dame by David Severson, professor of biological sciences. Decoding sand flies, ticks and several other mosquito species is also in the works.

Collins is confident that scientists will be able to genetically engineer the mosquito so diseases aren’t spread so easily. “Not only is it doable, it is in some respects a much more reasonable approach to dealing with problems of disease transmission,” he says. Much of Africa, where malaria looms largest, lacks the infrastructure to deliver modern medical solutions. “A lot of people still die of measles because we can’t get vaccine to them.”

If you can “improve” the mosquito, then harness this genetic legerdemain to a natural, infectious process, you don’t need infrastructure. Nature will finish the job.

Morphing mosquitoes

Two doors down from Collins’ office sits Nora Besansky, his wife and one of many scientific partners in this endeavor. On her door are taped two golden pipette awards, good-natured pokes from Notre Dame graduate students for being the most stressed out.

The biological sciences professor is trying to solve one of the most important riddles of mosquito ecology and malaria transmission. Anopheles gambiae is the predominant malarial vector in Africa, but it rarely works alone. Over eons, the species has evolved into those seven different identical-looking mosquitoes. In such West African nations as Burkina Faso and Mali, however, Anopheles gambiae appears to be evolving again, splitting into two forms known simply as M and S.

The traditional A. gambiae mosquito breeds in pools and puddles. It requires a rainy season, along with animals and people tromping through the mud, digging ditches, making bricks. During the dry season A. gambiae dies back, and malaria transmission subsides.

Twentieth-century engineering has brought a new habitat to West Africa, particularly in the thirsty southern semi-desert area of the Sahara known as the Sahel. Rice fields, irrigation ditches and impoundments are now spread over vast regions of previously arid land. Apparently Anopheles gambiae abhors a vacuum, because a subtle variant called M has begun to exploit this new habitat. Rice fields are flooded, and with two rice crops a year there is no more dry season. “There are greater numbers, and it’s found in places it never used to be found before and at times it never used to be found before,” says Besansky. More mosquitoes means more malaria.

Under a microscope it is impossible to tell the M form from S, but when scientists examine the sperm deposited in the females, it shows that the male and female match forms about 99 percent of the time. Even though the remaining 1 percent is enough to keep the species genetically mixed, Besansky believes they are on independent evolutionary tracks. Her mission is to find out how. There have to be genes underlying the mating preference, but it is difficult to study mating behavior. “They mate at dusk, in swarms,” she laughs. “Hello, you can’t do this in the lab. You can’t tell them apart.”

But the genomics toolbox can. A team at the University of California, Davis, transferred DNA from A. gambiae M and S to computer chips. A comparison showed three small regions of difference, and now Besansky and others are racing to decipher the details. That means going beyond the genome and back into the field. Her collaborators in Burkina Faso are doing the ecological work, trying to figure out how M and S interact on the ground. Funding from Besansky’s own grants and from the National Institutes of Health has helped build high-end malaria labs in Burkina Faso and neighboring Mali, which have been critical to the work. “This is a highly interdisciplinary project,” she says. “It would be impossible for me to do what I do without those good collaborators in Africa.”

The intricacy of the Anopheles gambiae complex, especially the M and S enigma, shows just how difficult even basic malarial biology can be. Will it be necessary to modify each species in the complex, or will it be enough to knock out the major human vectors, A. gambiae and its close relative, A. arabiensis? Or will other species fill the void? “Transgenic mosquitoes are not a magic bullet,” concludes Besansky. “There has to be a whole suite of tools. We are still going to need the insecticides, we are still going to need the bed nets. We’re going to need the antimalarial drugs and vaccines.”

Jumping genes

How do you make a transgenic mosquito?

No single mad scientist is likely to retreat to the lab to create one. Hundreds of investigators following dozens of lines of inquiry are engaged in this project. The paper enumerating the Anopheles gambiae genome, for example, has more than 100 authors.

For Malcom Fraser, Notre Dame professor of biological sciences, the quest began with a completely different organism. In the early 1980s Fraser was a graduate student working on baculoviruses, which inhabit insects and other invertebrates. His professor at Ohio State had noticed a mutation, a tiny change in the trail left by the virus. It suggested a little more DNA than expected. Fraser spent weeks at the microscope learning to spot the slight difference between the normal virus and the mutation.

Through years of fancy labwork, Fraser maintained his fascination with the mutation. Eventually he worked out the source of that extra DNA: It was a transposon, also known as a jumping gene. Transposons are virus-like segments of DNA that have the ability to move around, causing mutations. Most of the mutations are deleterious, but organisms have evolved ways to resist the influence of transposons. The resulting give-and-take can leave a hefty trail; some 45 percent of human DNA, for example, is thought to be made up of transposons and their inactive remains.

Because transposons move around so easily, geneticists have harnessed them to manipulate DNA. Fraser saw this potential and named the genetic sequence he had found piggyBac. He began exploring the transposon’s potential for re-engineering insects, with the ultimate goal of remaking the mosquito. By attaching a desirable gene to piggyBac—let’s say one that would frustrate the malaria parasite’s effort to enter the salivary glands of the mosquito—it could potentially deliver it into the mosquito. Even better, because of the infectious, viral behavior of transposons, they could perhaps penetrate a gene pool far faster than the simple laws of heredity would normally allow.

But the more species Fraser examined, the more evidence he discovered of piggyBac—or something very similar—all over the natural world. It suggested that at some point in evolutionary time piggyBac had moved between species. “That’s dangerous if you’re going to use them for transgenic engineering of insects that you intend to release in the field,” Fraser says. “These invasive DNAs can have a significant impact on new species when they get in and start jumping around and causing mutations. We’re talking about consequences that we can’t really predict. I don’t want to be known as the guy who released piggyBac and killed half the insects in a particular region of the world.”

There are other ways to engineer a mosquito, and Fraser is still working toward that goal. He’s received a Grand Challenge grant from the Bill and Melinda Gates Foundation to explore what he hopes will be a more stable method of genetic tinkering. Rather than tackle the monstrous complexity of the malaria parasite, he’s looking at dengue fever first. Dengue is a virus carried by the Aedes aegypti mosquito. It kills between 25,000 and 50,000 people a year, and in the last few decades a new and deadly hemorrhagic form has emerged.

Despite Fraser’s reluctance to use transposons, the method is by no means dead. Last spring Tony James at the University of California, Irvine, successfully used transposons to engineer a dengue-resistant mosquito. Severson, the Notre Dame professor who specializes in Aedes aegypti, will be working with James on the next stage of the project. The researchers hope to try large-scale cage trials in Mexico that would simulate ecological conditions in an endemic area.

No genetically modified mosquitoes will be released into the wild. “We are 10 or 20 years away from doing something like that, at least,” estimates Severson. The cage trials, however, can test both biological and social questions. Biologically, the scientists need to find out how the modified mosquito works outside the lab. Will it survive? Perhaps even more important, says Severson, is public awareness and education, and the ability of the scientists to work with the public and government agencies that will ultimately decide whether such a project should proceed.

“A large segment of the population has to be on board with this,” says Nora Besansky. What’s more, scientists have to find a way to make genetic modification work faster than the natural pace of evolution; otherwise it won’t seem practical. “I think we have to harness some of these genetic cheaters to make it happen in a frame that’s relevant for public health,” she says. “In fact, probably in a frame that’s relevant for a politician—five or 10 years.”

Besansky understands Fraser’s concern about transposons. “There are advantages and disadvantages to just about every one of the mechanisms that has been proposed,” she says.

Disease and development

Father Thomas G. Streit, CSC, ’80, ’85M.Div., ’94Ph.D. tells a more intimate tale of his work in Haiti, where he spends eight months of the year. Of the 65 employees he’s had under the age of 30, 10 have died. Several were lost to that nation’s general chaos and violence, but six have died of infectious diseases and one of prenatal eclampsia. None would have died in the United States. “These are people of some means because they had a job,” he says.

Streit works on lymphatic filariasis in Haiti. The parasite is also transmitted by mosquitoes and damages the human lymphatic system, causing swelling such as elephantiasis. The World Health Organization has targeted filariasis for elimination in Haiti by 2012. It’s a far easier task than malaria, but the planned solution shows just how tightly bound poverty and disease can be.

Filariasis can be eliminated through simple economic development. Its mosquito host breeds in raw sewage, so screens and basic investments in sanitation can accomplish a lot. But in Haiti even the simplest development goals can’t be taken for granted, so the preferred approach here is to fortify the salt supply with diethylcarbamazine, a drug that kills juvenile parasites. It’s the same idea as adding iodine to salt, but Streit found there was not even an iodization program in Haiti. Iodine is critical for brain development, and deficiencies can drop IQ by as much as 15 points. “Without adequate dietary iodine, these kids aren’t as smart as they should be,” says Streit. “Talk about handicaps to development! If we help them with these diseases, we’re going to give them a better chance to develop their country.”

For years, the disease and development dynamic has been a classic chicken-and-egg debate. Are some countries poor because of disease? Or is disease another symptom of poverty? In the end the problem cuts both ways, but that hasn’t stopped Columbia economist Jeffrey Sachs, director of the U.N. Millennium Project, from calling for a fundamental change in the economic strategies of wealthy nations toward the poor. In The End of Poverty, for example, Sachs examines the investment by rich nations in malaria and AIDS—diseases that particularly stunt African growth—and calls for “an end to the international community’s gross negligence regarding the diseases ravaging Africa.”

The Notre Dame biologists agree that simple economic development would go a long way toward eradicating disease, but Collins says development alone is not enough. “There are still some parts of the world where the intensity of infectious transmission of some of these diseases is so high that economics isn’t going to eliminate them,” he says. “There is still dengue in Singapore. Not a lot, but it’s there. You can’t get more economically well-developed and regulated than a place like Singapore.”

Meanwhile malaria in Africa enjoys an almost perfect storm of reinforcing factors. “The vectors are incredibly efficient,” says Collins. “Even putting screens in everybody’s houses isn’t going to stop people who are out of doors after dark from getting bitten. We’re not going to get rid of all the mosquitos in Africa just by economic development.”

Despite a festering public discomfort with genetically modified organisms, Collins thinks it’s important to remember that public health is a completely different motive than most of the agribusiness examples of genetic modification.

“Every 30 seconds someone dies from malaria, and the majority of those are children under the age of 5,” says David Severson. “Look a child in the eye in Western Kenya and say we can’t explore these options because we have issues with transgenic organisms. I think we have an obligation to pursue these things.”

Signs of hope

“I’ve been working in parasitology for 30 years. We’re worse off now than when I started,” says John Adams, Notre Dame professor of biological sciences. “People should be worried,” he adds, as he enumerates the landscape of disease from the resurgence of malaria to the accelerating emergence of new maladies such as AIDS and avian flu. “These are all signs that we’re not taking care of our environment in a way that’s conducive to good health.”

But there are also signs of hope. Sachs and other like-minded crusaders have put public health back on the map, leading to the Global Fund to Fight AIDS, Tuberculosis and Malaria. The Gates Foundation has energized the scientific community with a series of creative and effective grant programs.

New fields such as genomics are just beginning to reach their potential and spawn the kinds of serendipitous solutions the world needs. For example, Adams has spent years struggling with the virulent malarial parasite Plasmodium falciparum, which, despite its resilience in the wild, is extremely difficult to work with in the lab. It has been particularly resistant to the kinds of genetic manipulation typically used to unlock the final meaning of genes: what proteins they make.

But Fraser and Adams have discovered that piggyBac seems perfectly suited to breaking down falciparum. “With most things being difficult with falciparum, you couldn’t ask for more,” says Adams.

You could call it luck, but the discovery demonstrates just how collaborative research on these problems is, both within Notre Dame and beyond. “Even though we may be working on different diseases and different organisms, tropical diseases share many common features,” says Adams. “A whole group of people looking at different aspects of the same diseases are more likely to come up with novel ideas.”

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